Method of producing a taxane-type diterpene and a method of obtaining cultured cells which produce the taxane-type dilterpene at a high rate

ABSTRACT

This invention relates to a method of producing a taxane-type diterpene(s) wherein tissues or cells of a plant which produces taxane-type diterpene(s) is cultured in the presence of at least one selected from the group consisting of jasmonic acids, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents, a method of producing a taxane-type diterpene wherein the tissues or the cells of the plant are cultured by controlling the oxygen concentration in a gas phase in a culture vessel to less than the oxygen concentration in the atmosphere from the initial stage of the culture, or by controlling the dissolved oxygen concentration in a fluid medium which is in contact with the tissue or the cell to less than the saturated dissolved oxygen concentration at that temperature from the initial stage of the culture, a method of producing a taxane-type diterpene wherein the tissue or the cell of the plant is cultured in a culture vessel, while oxygenic gas containing 0.03-10% of carbon dioxide is used as aeration gas to be introduced to the vessel, and a method of obtaining highly productive cultured cells for the taxane-type diterpene wherein cultured cells of the plant which produces the taxane-type diterpene are separated into a plurality of layers according to the difference in their specific gravities, and the cells contained in at least one layer are cultured, then highly productive cultured cells for the taxane-type diterpene are selected from among those cultured cells.  
     The present invention allows industrial production of a taxane-type diterpene such as taxol which is useful as a therapeutic agent for cancer.

TECHNICAL FIELD

[0001] This invention relates to a method of producing a taxane-typediterpene including taxol which is useful as a therapeutic agent forovarian cancer, mammary cancer, lung cancer and the like, and a methodof obtaining cultured cells which produce the taxane-type diterpene at ahigh rate.

BACKGROUND ART

[0002] Taxol, which is useful as a therapeutic agent for ovarian cancer,mammary cancer, lung cancer and the like, is a taxane-type diterpeneidentified after being isolated from Taxus brevifolia NUTT, which is aplant belonging to genus Taxus, family Taxaceae and has a complex estergroup which is related to its activity. Taxol can be found in all theparts of the plant body of Taxus brevifolia NUTT, but the bark has beenreported to exceed all others in its content of the taxol. At present,taxol is collected from a natural or a cultivated plant body, however,the plant belonging to genus Taxus grows slowly, and it takes more than10 years to grow-to a height of 20 cm above the ground, besides the treedies after its bark is taken off, thus it has been difficult to easilyobtain a large amount of taxol. It would be advantageous if ataxane-type diterpene such as taxol and baccatin III which is aprecursor of taxol, can be produced by the use of tissue culture, sincea large amount of taxol can be easily obtained without cutting down thetrees.

[0003] As a conventional method of producing taxol by utilizing culturedplant cells, a U.S. patent was issued on a production method utilizingcultured cells of Taxus brevifolia NUTT (U.S. Pat. No. 5,019,504),however, the yield of taxol production described therein is 1-3 mg/l,and that is insufficient for the industrial production. Besides, theproduction of taxol by the cell culture is unstable and even when aprimary cell of high productivity can be obtained by selection, it isdifficult to keep its content by subculturing [E. R. M. Wickremesine etal., World Congress on Cell and Tissue Culture (1992)].

[0004] On the other hand, as a prior art in the taxol production, asemisynthetic method from baccatin III, which is a precursor inbiosynthesis of taxol, is disclosed in the specification of U.S. Pat.No. 5,015,744 issued to Holton et al. By the use of the plant tissueculture, a raw material for the semisynthetic process such as baccatinIII can be produced, thus the plant tissue culture can be also utilizedfor taxol production by the above-mentioned semisynthetic process.

DISCLOSURE OF THE INVENTION

[0005] The first object of the present invention is to provide a simplemethod of producing a taxane-type diterpene by plant tissue culture.

[0006] The second object of the present invention is to provide a methodof obtaining cultured cells which produce a taxane-type diterpene at ahigh rate.

[0007] The first invention of the present application is a method ofproducing a taxane-type diterpene wherein a tissue or a cell of a plantwhich produces a taxane-type diterpene is cultured in the presence of atleast one substance selected from the group consisting of jasmonicacids; compounds containing a heavy metal, complex ions containing aheavy metal, heavy metal ions, amines and antiethylene agents, then thetaxane-type diterpene is recovered from the resulting cultures.

[0008] The second invention of the present application is a method ofproducing a taxane-type diterpene wherein a tissue or a cell of a plantwhich produces a taxane-type diterpene is cultured by controlling theoxygen concentration in a gas phase in a culture vessel to less than theoxygen concentration in the atmosphere, from the initial stage of theculture, or by controlling the dissolved oxygen concentration in a fluidmedium which is in contact with the tissue or the cell to less than thesaturated dissolved oxygen concentration at that temperature, from theinitial stage of the culture, then the taxane-type diterpene isrecovered from the resulting cultures.

[0009] The third invention of the present application is a method ofobtaining cultured cells which produce a taxane-type diterpene at a highrate, wherein cells of a plant which produces a taxane-type diterpeneare fractionated into a plurality of layers according to the differencein their specific gravities, and cells contained in at least one layerare cultured, then such cultured cells that produce the taxane-typediterpene at a high rate are selected from among those cultured cells.

[0010] The present invention will be described in further detail.

[0011] The taxane-type diterpene, which is an object for the presentinvention, is not particularly limited to any diterpene as far as it hasa taxane skeleton, and the illustrative examples include taxol,7-epitaxol, baccatin III, 7-epibaccatin III, cephalomannine,7-epicephalomannine, 10-deacetylbaccatin III, 10-deacetylcephalomannine,10-deacetyltaxol, taxagifine, an analogue thereof, taxane la, ananalogue thereof, xylosyl cephalomannine, xylosyl taxol and the like.

[0012] Examples of the plant to be used in the present invention whichproduces the taxane-type diterpene are those belonging to genus Taxus,such as Taxus baccata LINN, Taxus cuspidata SIEB. et ZUCC, Taxuscuspidata SIEB. et ZUCC var. nana REHDER, Taxus brevifolia NUTT, Taxuscanadiensis MARSH, Taxus chinensis, and Taxus media.

[0013] According to the first invention of the present application,culture of the above-mentioned plant can be carried out by thepreviously known method except that the tissue or the cell of the plantwhich produces the taxane-type diterpene is cultured in the presence ofat least one substance selected from the group consisting of jasmonicacids, compounds containing a heavy metal, complex ions containing aheavy metal, heavy metal ions, amines and antiethylene agents.

[0014] Examples of jasmonic acids, which are objects for the firstinvention of the present application, include a compound represented bythe general formula (I):

[0015] [wherein, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e) and R^(1f)respectively represent hydrogen atom, hydroxyl group, alkyl group having1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms;

[0016] R², R³, R⁴, R⁵ and R^(6a) respectively represent hydrogen atom oralkyl group having 1 to 6′ carbon atoms; a side chain consisting ofC¹—C²—C³—C⁴—C⁵—C⁶ may contain one or more double bonds;

[0017] R^(6b) represents hydroxyl group or —O— carbohydrate residue;

[0018] R⁷ represents hydroxyl group, OM (wherein M is alkali metal atom,alkaline earth metal atom or NH₄), NHR⁸ (wherein R⁸ represents hydrogenatom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6carbon atoms or amino acid residue), OR⁹ (wherein R⁹ is alkyl grouphaving 1 to 6 carbon atoms or carbohydrate residue), or alkyl grouphaving 1 to 6 carbon atoms;

[0019] n is an integer of 1-7;

[0020] and in the above-mentioned five-membered ring, a double bond maybe formed between the neighboring member carbon atoms],

[0021] a compound represented by the general formula (II):

[0022] [wherein, R^(1e), R^(1b), R^(1c), R^(1d), R^(1a) and R^(1f)respectively represent hydrogen atom, hydroxyl group, alkyl group having1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms;

[0023] R², R³, R⁴, R⁵ and R⁶ respectively represent hydrogen atom oralkyl group having 1 to 6′ carbon atoms;

[0024] a side chain consisting of C¹—C²—C³—C⁴—C⁵—C⁶ may contain one ormore double bonds;

[0025] R⁷ represents hydroxyl group, OM (wherein M is alkali metal atom,alkaline earth metal atom or NH₄), NHR⁸ (wherein R⁸ represents hydrogenatom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6carbon atoms or amino acid residue), OR⁹ (wherein R⁹is alkyl grouphaving 1 to 6 carbon atoms or carbohydrate residue), or alkyl grouphaving 1 to 6 carbon atoms;

[0026] n is an integer of 1-7;

[0027] and in the above-mentioned five-membered ring, a double bond maybe formed between the neighboring member carbon atoms],

[0028] and a compound represented by the general formula (III):

[0029] [wherein, R^(1a), R^(1b), R^(1 c), R^(1d), R^(1e) and R^(1f)respectively represent hydrogen atom, hydroxyl group, alkyl group having1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms;

[0030] R², R³, R⁴, R⁵ and R⁶ respectively represent hydrogen atom oralkyl group having 1 to 6 carbon atoms;

[0031] a side chain consisting of C¹—C²—C³—C⁴—C⁵—C⁶ may contain one ormore double bonds;

[0032] R⁷ represents hydroxyl group, OM (wherein M is alkali metal atom,alkaline earth metal atom or NH₄), NHR⁸ (wherein R⁸ represents hydrogenatom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6carbon atoms or amino acid residue), OR⁹ (wherein R⁹ is alkyl grouphaving 1 to 6 carbon atoms or carbohydrate residue), or alkyl grouphaving 1 to-6 carbon atoms;

[0033] n is an integer of 1-7;

[0034] and in the above-mentioned five-membered ring, a double bond maybe formed between the neighboring member carbon atoms].

[0035] Preferable examples of jasmonic acids represented by theabove-mentioned general formula (I) include a compound represented bythe general formula (I′):

[0036] [wherein, R^(1′) represents hydrogen atom or hydroxyl group;

[0037] a side chain consisting of C¹—C²—C³—C⁴—C⁵—C⁶ may contain a doublebond between C¹ and C², between C² and C³, or between C³ and C⁴;

[0038] R^(6b) represents hydroxyl group or —O— carbohydrate residue;

[0039] R⁷ represents hydroxyl group, OM (wherein M is alkali metal atom,alkaline earth metal atom or NH₄), NHR^(8′) (wherein R^(8′) representshydrogen atom, acyl group having 1 to 4 carbon atoms, alkyl group having1 to 4 carbon atoms or amino acid residue) or OR⁹ (wherein R⁹ representsalkyl group having 1 to 4 carbon atoms or carbohydrate residue);

[0040] n is an integer of 1-7;

[0041] and in the above-mentioned five-membered ring, a double bond maybe formed between the neighboring member carbon atoms], and preferableexamples of jasmonic acids represented by the above-mentioned generalformula (II) include a compound represented by the general formula(II′):

[0042] [wherein, R¹ represents hydrogen atom or hydroxyl group; a sidechain consisting of C¹—C²—C³—C⁴—C⁵—C⁶ may contain a double bond betweenC¹ and C², between C² and C³, or between C³ and C⁴;

[0043] R^(7′) represents hydroxyl group, OM (wherein M is alkali metalatom, alkaline earth metal atom or NH₄), NHR^(8′) (wherein R^(8′)represents hydrogen atom, acyl group having 1 to 4 carbon atoms, alkylgroup having 1 to 4 carbon atoms or amino acid residue) or OR⁹ (whereinR^(9′) represents alkyl group having 1 to 4 carbon atoms or carbohydrateresidue);

[0044] n is an integer of 1-7;

[0045] and in the above-mentioned five-membered ring, a double bond maybe formed between the neighboring member carbon atoms], and preferableexamples of jasmonic acids represented by the above-mentioned generalformula (III) include a compound represented by the general formula(III′),

[0046] [wherein, R^(1′) represents hydrogen atom or hydroxyl group; aside chain consisting of C¹—C²—C³—C⁴—C⁵—C⁶ may contain a double bondbetween C¹ and C², between C² and C³, or between C³ and C⁴; R^(7′)represents hydroxyl group, OM (wherein M is alkali metal atom, alkalineearth metal atom or NH₄), NHR^(8′) (wherein R^(8′) represents hydrogenatom, acyl group having 1 to 4 carbon atoms, alkyl group having 1 to 4carbon atoms or amino acid residue) or OR^(9′) (wherein R^(9′)represents alkyl group having 1 to 4 carbon atoms or carbohydrateresidue);

[0047] n is an integer of 1-7;

[0048] and in the above-mentioned five-membered ring, a double bond maybe formed between the neighboring member carbon atoms].

[0049] In the above-mentioned general formulae (I), (II) and (III),examples of alkyl group having 1 to 6 carbon atoms represented byR^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵, R⁶,R^(6a), R⁷, R⁸ or R⁹ include, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, and n-hexylgroups.

[0050] In the above-mentioned general formulae (I), (II) and (III),examples of alkoxy group having 1 to 6 carbon atoms represented byR^(1a), R^(1b), R^(1c), R^(1d), R^(1e) or R^(1f) include, for example,methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy,t-butoxy, n-pentyloxy and n-hexyloxy groups.

[0051] When R⁷ is OM, examples of an alkali metal atom or an alkalineearth metal atom represented by M include, for example, sodium,potassium and calcium.

[0052] When R⁷ is NHR⁸, the acyl group having 1 to 6 carbon atomsrepresented by R⁸ may have either a straight chain or a branched chain,and their examples include, for example, formyl, acetyl, propionyl,butyryl, valeryl, hexanoyl and acryloyl groups. When R⁷ is NHR⁸,examples of an amino acid residue represented by R⁸ include isoleucyl,tyrosyl, and tryptophyl groups.

[0053] When R⁷ is OR⁹, an example of a carbohydrate residue representedby R⁹ is glucopyranosyl group, and when R^(6b) is —O— carbohydrateresidue in the above-mentioned general formula (I), an example of acarbohydrate residue is glucopyranosyl group.

[0054] In the compounds represented by the general formulae (I), (II)and (III), a double bond may be formed between the neighboring membercarbon atoms in the five-membered ring.

[0055] Illustrative examples of the compound represented by the generalformula (I) include those shown as follows;

[0056] Illustrative examples of the compound represented by the generalformula (I) include those shown as follows;

[0057] general formula (II) include those shown as follows;

[0058] Illustrative examples of the compound represented by the generalformula (III) include those shown as follows;

[0059] (Compound I)

[0060] R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵,R⁶:H

[0061] A double bond is formed between C³ and C⁴.

[0062] R⁷: —OH or —OCH₃

[0063] n: 1 to 3

[0064] (Compound J)

[0065] R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵,R⁶: H

[0066] R⁷: —OH

[0067] n: 1

[0068] Illustrative examples of the compound represented by the generalformula (III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e) or R^(1f)is hydroxyl group, or a double bond is formed between the neighboringmember carbon atoms in the five-membered ring, include those shown asbelow;

[0069] Preferable examples of a compound represented by the generalformula (II) or (III) include the compounds wherein R^(1a), R^(1b),R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R¹⁴, R⁵ and R⁶ are hydrogenatoms, R⁷ is hydroxyl group or methoxy group, and a side chainconsisting of C¹—C²—C³—C⁴—C⁵—C⁶ does not contain a double bond, orcontain a double bond between C¹ and C², between C² and C³, or betweenC³ and C⁴.

[0070] Jasmonic acids to be used in the present invention which arerepresented by the general formula (I), (II) or (III) have variousstereoisomers (cis-trans isomers and optical isomers), and each isomercan be used alone or in the form of a mixture. All of the jasmonic acidsshown above have the effect of improving the productivity in thetaxane-type diterpene production, however, tuberonic acid, methyltuberonate, cucurbic acid, methyl cucurbate, jasmonic acid and methyljasmonate, which are the compounds represented by the general formula(I), (II) or (III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e),R^(1f), R², R³, R⁴, R⁵ and R⁶ are hydrogen atoms, R⁷ is hydroxyl groupor methoxy group, n is 1, and a double bond is formed between C³ and C⁴,are particularly preferable from the view point of their higheffectiveness in improving the productivity.

[0071] These jasmonic acids are prepared by synthesis or extraction andthe like from a plant (H. Yamane et al: Agric. Biol. Chem., 44,2857-2864(1980)).

[0072] By the way, there is a description teaching that jasmonic acidsare produced by various plants by themselves as a phytohormone-likesubstance which induces various reactions related to growth promotion,maturation of tissue and appearance of resistance to disease (TeruhikoYoshihara. Shokubutsu Saib{overscore (o)} K{overscore (o)}gaku, Vol 2,No.4 523-531 (1990)).

[0073] Accordingly, the jasmonic acids involved in the presentinvention, can be not only added from outside of the culture system, butalso produced by the cultured cells or cultured tissues by themselves.An example of a method to promote the production of such endogenousjasmonic acids by the cultured cells or cultured tissues includesaddition of microorganism cultures, an extract or a heat-treatedsubstance thereof, or a plant extract to a culture medium, and anillustrative example of such a method is a process of adding a funguscell wall fraction described by M. J. Mueller et al., Proc. Natl. Acad.Sci. U.S.A., 90 (16), 7490-7494 (1993)). It is also possible to increasethe amount of the produced endogenous jasmonic acid by partiallydamaging cultured cells or cultured tissues mechanically, or withultraviolet rays, dr heat, and one illustrative example of such aprocess is mechanical cytoclasis of a part of cells (R. A. Cleeman etal., Proc. Natl. Acad. Sci. U.S.A., 89(11), 4938-4941 (1989).

[0074] Since jasmonic acids are hardly soluble in water, they areusually dissolved in an organic solvent such as ethanol and methanol ordissolved in a surfactant and the like, then added to a culture medium.Jasmonic acids in liberated form can be used as they are, or they areused in the form of a salt by being neutralized with an alkali.

[0075] Of the jasmonic acids, those compounds represented by the formula(I) or (III) tend to be in stable trans-form rather than unstablecis-form, since epimerization occurs at the alpha-position to thecarbonyl group in the five-membered ring by an acid, an alkali or heat.In an equilibrium experiment utilizing natural or synthesized jasmonicacids, the trans-form is present in the ratio of 90% and the cis-form ispresent in the ratio of 10%. Generally, cis-form is considered to have ahigher activity, but jasmonic acids to be used in the present inventioninclude all the stereoisomers of the compounds represented by theabove-mentioned formula (I) or (III), and the mixture thereof.

[0076] Jasmonic acids are required to have a concentration in a culturemedium of 0.01-1000 μM, and it is particularly preferable, according tothe first invention of the present application, to control theconcentration of the jasmonic acids to be in the range of 0.1 to 500 μM.

[0077] Induction of some secondary metabolites by addition of jasmonicacids to plant cell cultures is described in DE 4122208 C1, however,there have been no reports on carrying out tissue culture of ataxane-type diterpene producing plant in the presence of jasmonic acidsas a medium additive, and it has been beyond all expectations that theamount of the produced taxane-type diterpene, which has totallydifferent biosynthetic pathway or biosynthesis controlling mechanismfrom those of the secondary metabolites disclosed in the above-mentionedpatent, was increased by the method of the first invention of thepresent application.

[0078] There is a description in the International Publication WO No.93/17121 that jasmone or methyl jasmone, which has a structure analogousto those of jasmonic acids represented by the formula (I), (II) or (III)to be used in the present invention, is effective in induction of taxolproduction. However, these compounds do not have a group such ascarboxyl, which is represented by the formula: —-(CH₂)_(n)—CO—R⁷ in theformula (I), (II) or (III), unlike the jasmonic acids, and the taxolinducing activity of these compounds was found to be low (seeComparative Example No. 24).

[0079] The heavy metals, which are objects for the first invention ofthe present application, are not particularly limited to any heavy metalas far as it belongs to the copper group or the iron group, however, asthe metal belonging to the copper group, the use of silver isparticularly preferable, and as the metal belonging to the iron group,the use of cobalt is particularly preferable. In addition to that, whensilver or cobalt is used, it is preferably used in the form of acompound containing the said heavy metal, a complex ion containing thesaid metal or in the form of the said metal ion. These compounds can beused alone or in combination.

[0080] Illustrative examples of the compound containing silver includesilver nitrate, silver sulfate, silver fluoride, silver chlorate, silverperchlorate, silver acetate, silver sulfite, silverhexafluorophosphate(V), silver tetrafluoroborate, diamine silver(I)sulfate, potassium diaminoargentate(I) and the like. Among these,particularly preferable compounds can be exemplified by silver nitrate,silver sulfate and the like.

[0081] Illustrative examples of the complex ion containing silverinclude [Ag(S₂O₃)₂]³⁻, [Ag(S₂O₃)₃]⁵⁻, [Ag(NH₃)₂]⁺, [Ag(CN)₂]⁻,[Ag(CN)₃]²⁻, [Ag(SCN)₂]⁻, [Ag(SCN)₄]³⁻ and the like. Among these,particularly preferable complex ions can be exemplified by[Ag(S₂O₃)₂]³⁻, [Ag(S₂O₃)₃]⁵⁻ and the like.

[0082] Illustrative examples of the compound containing cobalt includecobalt chloride, cobalt nitrate, cobalt sulfate, cobalt fluoride, cobaltperchlorate, cobalt bromide, cobalt iodide, cobalt selenate, cobaltthiocyanate, cobalt acetate, ammonium cobalt sulfate, cobalt(II)potassium sulfate, hexaamminecobalt(III) chloride,pentaammineaquacobalt(III) chloride, nitropentaamminecobalt(III)chloride, dichlorotetraamminecobalt(III) chloride hemihydrate,dinitrotetraamminecobalt(III) chloride, carbonatotetraamminecobalt(III)chloride, ammonium tetranitrodiamminecobaltate(III), sodiumhexanitrocobaltate(III), tris(ethylenediamine)cobalt(III) chloridetrihydrate, dichlorobis(ethylenediamine)cobalt(III) chloride, potassiumtris(oxalato)cobaltate(III) trihydrate, potassiumhexacyanocobaltate(III), potassium(ethylenediaminetetraacetato)cobaltate(III) dihydrate,hydridotetracarbonylcobalt(I), dicarbonyl(cyclopentadienyl)cobalt(I),octacarbonyldicobalt(O), hexacarbonyl(acetylene)dicobalt(O),bis(cyclopentadienyl)cobalt(I),(cyclopentadienyl)(1,5-cyclooctadiene)cobalt(I) and the like. Amongthese, particularly preferable compounds can be exemplified by cobaltchloride, cobalt nitrate, cobalt sulfate and the like.

[0083] Illustrative examples of the complex ion containing cobaltinclude pentaammineaquacobalt ion, nitropentaamminecobalt ion,dichlorotetraamminecobalt ion, dinitrotetraamminecobalt ion,carbonatotetraamminecobalt ion, tetranitrodiamminecobalt ion,hexanitrocobalt ion, tris(ethylenediamine)cobalt ion,dichlorobis(ethylenediamine)cobalt ion, tris(oxalato)cobalt ion,hexacyanocobalt ion, (ethylenediaminetetraacetato)cobalt ion and thelike.

[0084] Of the said heavy metals, the compound containing silver, thecomplex ion containing silver or the silver ion preferably has aconcentration in the medium of 10⁻⁸M-10⁻¹M, and it is further preferableto adjust the concentration to be in the range of 10⁻⁷M to 10⁻²M. Thecompound containing cobalt, the complex ion containing cobalt or thecobalt ion preferably has a concentration in the medium of 10⁻⁶-10⁻¹M,and it is further preferable to adjust the concentration to be in therange of 10⁻⁵ to 10⁻²M.

[0085] So far, there are no cases reported wherein the tissue culture ofa plant producing a taxane-type diterpene is carried out in the presenceof a compound containing silver, a complex ion containing silver orsilver ion as an additive to the medium. Although compounds containingcobalt, or cobalt ions are contained as one of the medium components forsuch a medium that is generally used as a medium for the tissue cultureof a plant belonging to genus Taxus, such as Linsmaier-Skoog medium,Murashige-Skoog medium, and Gamborg's B-5 medium, they are used at aconcentration of 1 ×10⁻⁷M -4×10⁻⁷M [Growth and breeding of a woodyplant, edited by the latest biotechnology complete works editorscommittee, Nogyo Tosho, P265-268], which is a much lower concentrationthan those used in the method of the present invention. In the meantime,there are no cases reported wherein the tissue culture of a plantproducing a taxane-type diterpene is carried out in the presence of acompound containing cobalt or cobalt ions of such a high concentrationthat is used in the first invention of the present application, justlike the case with the above-mentioned silver compound. In addition tothat, it was beyond all expectations that the amount of the taxane-typediterpene to be produced is increased by the culture carried out in thepresence of such heavy metals.

[0086] According to the first invention of the present application, byamines we refer to an amine or a salt thereof. As the amines, which arethe objects for the first invention of the present application, bothmonoamines and polyamines can be used, however, the use of polyamines isparticularly preferable.

[0087] In addition to that, examples of the amines, which are theobjects for the first invention of the present application, includemono, di or trialkyl amines wherein a part of hydrogen atoms in thealkyl group may be substituted by hydroxyl group, such as methyl amine,ethyl amine, dimethyl amine, diethyl amine, triethyl amine, diethanolamine, triethanol amine or a salt thereof; polymethylene diamine whereinthe polymethylene moiety may be interrupted by imino group, and H in theamino group can be substituted by lower alkyl group, such as putrescine,cadaverine, spermidine, spermin, ethylenediamine,N,N-diethyl-1,3-propane diamine, triethylene tetramine, or a saltthereof; cyclic alkyl amine such as cyclopentyl amine, cyclohexyl amineor a salt thereof, or a cyclic amine such as methenamine and piperazine,or a salt thereof. Among these amines, preferable amines can beexemplified by polyamines such as putrescine [NH₄(CH₂)₄NH₂], cadaverine[NH₂(CH₂)₅NH₂], spermidine [NH₂(CH₂)₃NH(CH₂)₄NH₂], spermin[NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂], ethylene diamine [NH₂(CH₂)₂NH₂],N,N-diethyl-1,3-propane diamine [(C₂H₅)₂N(CH₂)₃NH₂], diethylene triamine[NH₂(CH₂)₂NH(CH₂)₂NH₂] and the like or a salt thereof.

[0088] The said amines preferably have a concentration in the medium of10⁻⁸M-10⁻¹M, and it is further preferable to adjust the concentration tobe in the range of 10⁻⁷M to 10⁻²M.

[0089] One illustrative example wherein a secondary metabolite is shownto be induced by addition of amines to the plant tissue cultures isshown in Japanese Patent Laid-Open Publication No. 4-262788 whereinindole alkaloid production is shown to be induced by addition of aminesto cultured cells of Catharanthus roseus. However, there are no casesreported wherein the tissue culture of a plant producing taxane-typediterpene, which is a different plant species from that of Catharanthusroseus, was carried out in the presence of amines as an additive to themedium, and it was beyond all expectations that the amount of thetaxane-type diterpene, which has a totally different biosyntheticpathway from that of the indole alkaloid, to be produced can beincreased thereby.

[0090] The antiethylene agent, which is an object for the firstinvention of the present application is not particularly limited to anyspecific substance as far as it is a substance which inhibits theethylene biosynthesis mechanism of the cultures and/or a substance whichremoves the ethylene remaining in the cultures or existing in the gasphase or in the medium in the culture vessel containing the cultures.

[0091] Illustrative examples of a method to inhibit the ethylenebiosynthesis mechanism include a method of inhibiting the activity of anenzyme which catalyzes the conversion of S-adenosyl-methionine into1-aminocyclopropane-1-carboxylic acid, and a method of inhibiting theactivity of an enzyme which catalyzes the conversion ofl-aminocyclopropane-1-carboxylic acid into ethylene, and illustrativeexamples of the compound having the former function include,aminoxyacetic acid, acetylsalicylic acid, Rhizobitoxine,aminoethoxyvinylglycine, methoxyvinylglycine, a-aminoisobutyric acid,2,4-dinitrophenol and the like. They can also include a salt, an ester,an amino acid derivative and a carbohydrate derivative of the saidcompound.

[0092] Illustrative examples of the salt include sodium, potassium,calcium, and magnesium salts, illustrative examples of the ester includemethyl, ethyl, propyl, and butyl esters, illustrative examples of theamino acid derivatives include glycine, methionine, and phenylalaninederivatives, and illustrative examples of the carbohydrate derivativeinclude glucose and maltose derivatives. The salt, ester, amino acidderivative, carbohydrate derivative according to the present inventionare not limited to the above-mentioned compounds.

[0093] Illustrative examples of the compound having the latter functioninclude gallic acid, a salt, an ester, an amino acid derivative and acarbohydrate derivative thereof [Hiroshi Hyodo, Society of HorticultureAutumn Convention 1987 Symposium Summary, p. 122, Susumu Kuraishi,Phytohormone, Tokyo University Publication, p.111].

[0094] Illustrative examples of the salt include sodium, potassium,calcium, and magnesium salts, illustrative examples of the ester includemethyl, ethyl, propyl, and butyl esters, illustrative examples of theamino acid derivatives include glycine, methionine, and phenylalaninederivatives, and illustrative examples of the carbohydrate derivativesinclude glucose and maltose derivatives. The salt, ester, amino acidderivative, carbohydrate derivative according to the present inventionare not limited to the above-mentioned compounds.

[0095] Illustrative examples of the substance which removes the ethyleneremaining in the cultures or existing in the gas phase or the medium inthe culture vessel containing the cultures include 1,5-cyclooctadieneand isothiocyanic acid, a salt, an ester (such as allyl isothiocyanateand benzyl isothiocyanate), an amino acid derivative and a carbohydratederivative thereof [Megumi Munakata, Chemical control in plants, 29(1),89-93 (1994)]. Illustrative examples of the salt include sodium,potassium, calcium, and magnesium salts, illustrative examples of theester include methyl, ethyl, propyl, butyl, and allyl esters,illustrative examples of the amino acid derivatives include glycine,methionine, and phenylalanine derivatives, and illustrative examples ofthe carbohydrate derivatives include glucose and maltose derivatives.The salt, ester, amino acid derivative, carbohydrate derivativeaccording to the present invention are not limited to theabove-mentioned compounds.

[0096] The antiethylene agent is required to have a concentration in aculture medium of 10⁻⁸M-10⁻¹M, and it is particularly preferable tocontrol the concentration of the antiethylene agent to be in the rangeof 10⁻⁷M to 10⁻²M.

[0097] It is known that ethylene is one of phytohormones, and involvedin various physiological phenomena caused in the plant, such as growthof individium, morphogenesis, and aging. A report by Kim, Dong II etal., Biotechnol. Bioeng., 38(4), 331-339 (1991) is an illustrativeexample wherein ethylene is utilized for improving the productivity ofthe secondary metabolite by the plant. However, -in all the exampleswherein controlling of ethylene is utilized for improving theproductivity of the secondary metabolite, it is the control of ethylenesupply to the plant tissue cultures, as typically shown in theabove-mentioned report, and so far there have been no cases reported inwhich the control to inhibit the ethylene production is utilized toimprove the production of the secondary metabolite, like the method ofthe present invention.

[0098] In addition to that, the antiethylene agent is generally utilizedas a freshness keeping agent for flowers, fruits and vegetables,however, there have been no cases reported wherein the antiethyleneagent is used for the purpose of improving the production of thesecondary metabolite.

[0099] Under these circumstances, the present inventors ascertained thatethylene greatly inhibits the production of the taxane-type diterpene bythe tissues and the cells of the taxane-type diterpene producing plant.Accordingly, based on the above-mentioned finding, the inventorscultured the said tissue cultures in the presence of the antiethyleneagent, and found out that the antiethylene agent not only controls theabove-mentioned inhibition but also remarkably improves the amount ofthe taxane-type diterpene resulting from the cultures. There have beenno cases reported wherein the production of the taxane-type diterpene isinduced by culturing the tissue cultures of a plant producingtaxane-type diterpene in the presence of an antiethylene agent, and itwas beyond all expectations that the productivity of the above-mentionedsecondary metabolite can be even increased by the method of the firstinvention of the present application.

[0100] Examples of the medium to be used for the first invention of thepresent application include those known media which have beenconventionally used for the plant tissue culture, such as medium ofMurashige & Skoog (1962), medium of Linsmaier Skoog (1965), Woody PlantMedium (1981), Gamborg's B-5 medium and Mitsui's M-9 medium.

[0101] A phytohormone, and if necessary a carbon source, an inorganiccomponent, vitamins, amino acids and the like may be added as well tothese media.

[0102] As a carbon source, a disaccharide such as sucrose, maltose, andlactose, a monosaccharide such as glucose, fructose and galactose,starch or a mixture of two or more kinds of such sugar sources mixed atan appropriate ratio can be utilized.

[0103] As an inorganic component, illustrative examples includephosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron,manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine andcobalt, and these components can be added in the form of such a compoundas potassium nitrate, sodium nitrate, calcium nitrate, potassiumchloride, potassium monohydrogenphosphate, potassiumdihydrogenphosphate, calcium chloride, magnesium sulfate, sodiumsulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zincsulfate, boric acid, copper sulfate, sodium molybdate, molybdenumtrioxide, potassium iodide, cobalt chloride and the like.

[0104] As the phytohormone, for example, auxin such as indoleacetic acid(IAA), naphthalenacetic acid (NAA), and 2,4-dichlorophenoxy acetic acid(2,4-D), and cytokinin such as kinetin, zeatin, and dihydrozeatin can beused.

[0105] As the vitamins, for example, biotin, thiamin (vitamin B1),pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid andthe like can be used.

[0106] As the amino acids, for example, glycine, phenylalanine, leucine,glutamine, cysteine and the like can be added.

[0107] Generally, the carbon source in a concentration of about 1-about30 g/l, the inorganic component in a concentration of about 0.1 μM-about100 mM, the phytohormones in a concentration of about 0.01-about 10 μM,and the vitamins and the amino acids respectively in a concentration ofabout 0.1-about 100 mg/l are used.

[0108] According to the present invention, both a liquid medium and sucha solid medium that contains agar and gelan gum normally in an amount of0.1-1% can be used, however, usually a liquid medium is preferable.

[0109] According to the tissue-culture of the present invention, a pieceof a tissue or a cell of a root, a growing point, a leaf, a stem, aseed, a pollen, an anther and a calyx and the like of the said plant orcultured cells which are obtained by the tissue culture thereof in theabove-mentioned medium or another conventional medium can be used.

[0110] The present invention can also be applied to neoplastic celland/or hairy-root, obtained by infecting a plant tissue withAgrobacterium tumefaciens or Agrobacterium rhizogenes.

[0111] By culturing these tissues or cells in the presence of at leastone substance selected from the group consisting of jasmonic acids,compounds containing a heavy metal, complex ions containing a heavymetal, heavy metal ions, amines, and antiethylene agents, culturedtissues or cultured cells having higher taxane-type diterpeneproductivity than that of those obtained by the tissue culture carriedout under the normal culture conditions, can be obtained. When at leastone compound selected from compounds containing a heavy metal, complexions containing a heavy metal, heavy metal ions, amines, andantiethylene agents is used together with jasmonic acids represented bythe above-mentioned general formulae (I), (II) or (III), the effect ofthe first invention of the present application can be enhanced.

[0112] Taxane-type diterpene can be fractionated from the cultures suchas cultured tissues, cultured cells and culture medium, which areobtained according to the above-mentioned process, by extraction with anorganic solvent such as methanol. It is also possible to recover thetaxane-type diterpene continuously during culture by allowing anappropriate adsorbing agent or an organic solvent coexist in the culturemedium.

[0113] One preferable example of the tissue culture according to thepresent invention can be illustrated as follows.

[0114] A piece of a plant body of a plant belonging to genus Taxus, suchas a root, a growing point, a leaf, a stem, a seed and the like issterilized and placed on Woody Plant Medium solidified with gelan gum,and kept at 10-35° C. for about 14-60 days so that a part of the tissuepiece is changed to callus. By subculturing the callus thus obtained,the growing speed is gradually increased and stabilized callus can beobtained. By the stabilized callus, we refer to a callus which remainsin callus state during culture without showing differentiation into ashoot or a root and the cells of which have uniform growing speed.

[0115] Such stabilized callus is transferred to a liquid medium, suitedfor the growth, such as liquid Woody Plant Medium and grown. The growingspeed is further increased in the liquid medium. According to thepresent invention, the stabilized callus or the cells constituting theabove-mentioned callus are grown in a solid medium or a liquid medium inthe presence of at least one substance selected from a group consistingof jasmonic acids, compounds containing a heavy metal, complex ionscontaining a heavy metal, heavy metal ions, amines and antiethyleneagents. And, it is also possible to fractionate the stabilized callus orthe cells constituting the said callus into a plurality of layersaccording to the difference in their specific gravities and grow thecells contained in at least one layer in a culture medium containing atleast one substance selected from the group consisting of jasmonicacids, compounds containing a heavy metal, complex ions containing aheavy metal, heavy metal ions, amines and antiethylene agents.

[0116] In a generally known method to fractionate the cells according totheir specific gravities, density gradient is formed by a medium forcentrifugal separation, and the cells are layered over it, thencentrifugal separation is carried out.

[0117] As a medium for centrifugal separation, Ficoll, Percoll (bothproduced by Pharmacia LKB Biotechnology Co. Ltd.,), sucrose and cesiumchloride and the like are used. In the examples including Example No.5,the density gradient was produced by the use of Ficoll, however, themedium is not particularly limited to any substance as far as it doesnot damage the cells.

[0118] The number of the layers forming the density gradient is notparticularly restricted. The difference between the specific gravitiesof layers is not particularly limited and each difference in thespecific gravity can be the same or different.

[0119] Accordingly, the definition of the density gradient includes acase wherein the gradient changes continuously (the condition whereinthe number of the layers forming the density gradient is close toinfinite, and the specific gravity difference between each layer isclose to 0).

[0120] The cells can be fractionated into a plurality of layersaccording to the difference in their specific gravities by thus formingthe density gradient, layering the cells and carrying out thecentrifugal separation.

[0121] The specific gravity of the layer to be formed is normally in therange of 1.00 to 1.20 g/ml, preferably in the range of 1.03 to 1.11g/ml. As a layer to become an object for culture, at least one layer isselected, but it is also possible to select all the layers and culturethem.

[0122] When a plurality of layers are selected and cells contained inthe selected layers are cultured, it is possible to culture the cells inthese layers individually, but, it is also possible to mix the cells intwo or more layers of the selected plurality of layers and culture them.

[0123] The cultured cells having high taxane-type diterpene productivitycan be usually obtained by culturing cells contained in a layer havingthe specific gravity of 1.07 or less, but it is not always limited tothis range, since it may fluctuate depending on the cells to be culturedor the culture conditions. There is also a tendency that the cells in alayer of a higher specific gravity, have a higher content of thetaxane-type diterpene at the time when the fractionation is carried outaccording to the difference in the specific gravities. Accordingly, toensure that cultured cells which produce the taxane-type diterpene at ahigh rate can be obtained, it is desirable that the cells in all thefractionated layers are cultured for a certain period, then theconcentration of the taxane-type diterpene in the cells of each layer ismeasured, and the layer containing the cultured cells which produce thetaxane-type diterpene at a high rate is selected from among thoselayers.

[0124] It is also possible to fractionate the cultured cells into aplurality of layers according to the difference in the specificgravities by preparing a medium for centrifugal separation having oneparticular specific gravity such as 1.07 g/ml, for example, and carryingout the centrifugal separation according to the above-mentioned method.

[0125] Furthermore, the first invention of the present application canbe used together with the method of the second invention of the presentapplication wherein the culture is carried out by controlling the oxygenconcentration in a gas phase in a culture vessel to less than the oxygenconcentration in the atmosphere, from the initial stage of the culture,or by controlling the dissolved oxygen concentration in a fluid mediumwhich is in contact with the tissue or the cell to less than thesaturated dissolved oxygen concentration at that temperature from theinitial stage of the culture.

[0126] Here, by the initial stage of the culture, we refer to from thetime when the culture was started through the 7th day after the start ofthe culture, and the controlling of the oxygen concentration in the gasphase in the culture vessel or the controlling of the dissolved oxygenconcentration in the fluid medium which is in contact with the tissue orthe cell is preferably done from the beginning of the culture Thecontrolling period is not particularly limited, and, the controllingunder the said conditions can be done in the entire culture period, oronly in a part of the entire culture period, however, it is preferableto carry out the control at least for 3 days during the entire cultureperiod.

[0127] The oxygen concentration in the gas phase in the culture vesselis required to be controlled to 4-15%, and it is particularly preferableto control it to 6-12%. The dissolved oxygen concentration in the fluidmedium is required to be controlled to 1-75% of the saturated dissolvedoxygen concentration at that temperature and it is particularlypreferable to control it to 10-75%.

[0128] It is also possible to combine the first invention of the presentapplication, the second invention of the present application and thethird invention of the present application all together.

[0129] According to the first invention of the present application, itis effective to add jasmonic acids when the cultured cells are in theexponential growth phase or in the stationary phase, and it isparticularly preferable to add jasmonic acids in a transitional periodfrom the exponential growth phase to the stationary phase. The same canbe said of the timing of the treatment for increasing the amount of theendogenous jasmonic acids to be produced. For example, when cells aresubcultured in every 21 days, the 7th-16th day is the suitable time foraddition of the jasmonic acids or the treatment to increase the amountof the endogenous jasmonic acids to be produced, and when the cells inthe exponential growth phase, for example those on the 7th-14th day areto be subcultured, the suitable time is immediately after thetransplantation. The addition of the jasmonic acids or the treatment toincrease the amount of the endogenous jasmonic acid to be produced canbe done at a time, in a plurality of parts, or continuously.

[0130] It is effective to add compounds containing a heavy metal,complex ions containing a heavy metal or heavy metal ions after thebeginning of the culture and before the transitional period of thecultured cells from the exponential growth phase to the stationaryphase, and it is particularly preferable to add them at the beginning ofthe culture. The addition of the said compounds or the ions can be doneat a time, or in a plurality of parts.

[0131] It is effective to add amines before the transitional period ofthe cells from the exponential growth phase to the stationary phase, andit is particularly preferable to add them at the beginning of theculture. The addition of said compounds can be done at a time or in aplurality of parts.

[0132] It is effective to add antiethylene agents before thetransitional period of the cells from the exponential growth phase tothe stationary phase, and it is particularly preferable to add themimmediately after the transition to the stationary phase. The additionof the said compounds can be done at a time or in a plurality of parts.

[0133] The temperature for the tissue culture according to the firstinvention of the present application is usually about 10-about 35° C.,and preferably about 23-28° C. according to the high growing speed. Asfor the culture period, 14-42 days are preferable.

[0134] When a liquid medium is used for the culture according to thefirst invention of the present application, the cultured cells can befractionated from the culture medium after the culture is completed, bysuch a method as decantation or filtration and the desired taxane-typediterpene can be fractionated from the cultured cells and/or the culturemedium by such a method as extraction with an organic solvent

[0135] The second invention of the present application will be explainedas follows.

[0136] According to the second invention of the present application, theculture of the plant means culture of a tissue or a cell of the plant,wherein the culture is carried out by a conventionally known processexcept that the culture is carried out by controlling the oxygenconcentration in the gas phase of the culture vessel to below theatmospheric oxygen concentration from the initial stage of the culture,or by controlling the dissolved oxygen concentration in the fluid mediumwhich is in contact with the tissue or the cell to below the saturateddissolved oxygen concentration at that temperature from the initialstage of the culture.

[0137] So far, in the culture of a plant producing the taxane-typediterpene, there has been no reports wherein the culture is carried outunder such conditions that the oxygen concentration in the gas phase tobe supplied to the culture vessel wherein the tissue or the cells arecultured or the dissolved oxygen concentration in the medium which is incontact with the tissue or the cells to below the atmospheric oxygenconcentration or below the saturated dissolved oxygen concentration, andit was beyond all expectations that the amount of the taxane-typediterpene to be produced is increased by that.

[0138] According to the second invention of the present application, theoxygen concentration in the gas phase of the culture vessel wherein thetissue or the cells are cultured is required to be controlled to 4-15%,it is particularly preferably controlled to 6-12%. The dissolved oxygenconcentration of the fluid medium which is in contact with the tissue orthe cells is required to be controlled to 1-75% of the saturateddissolved oxygen concentration at that temperature, it is particularlypreferably controlled to 10-75%.

[0139] Examples of a medium to be used in the second invention of thepresent application, include the medium conventionally known for thetissue culture of a plant, such as medium of Murashige & Skoog (1962),medium of Linsmaier Skoog (1965), Woody Plant Medium (1981), Gamborg'sB-5 medium, and Mitsui's M-9 medium and the like.

[0140] A phytohormone, and if necessary a carbon source, an inorganiccomponent, vitamins, amino acids and the like may be added as well tothese media.

[0141] As a carbon source, a disaccharide such as sucrose, maltose, andlactose, a monosaccharide such as glucose, fructose and galactose,starch or a mixture of two or more kinds of such sugar sources mixed atan appropriate ratio can be utilized. As an inorganic component,illustrative examples include phosphorus, nitrogen, potassium, calcium,magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum,chlorine, sodium, iodine and cobalt, and these components can be addedin the form of such a compound as potassium nitrate, sodium nitrate,calcium nitrate, potassium chloride, potassium monohydrogenphosphate,potassium dihydrogenphosphate, calcium chloride, magnesium sulfate,sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zincsulfate, boric acid, copper sulfate, sodium molybdate, molybdenumtrioxide, potassium iodide, cobalt chloride and the like.

[0142] As the phytohormone, for example, auxin such as indoleacetic acid(IAA), naphthalenacetic acid (NAA), and 2,4-dichlorophenoxy acetic acid(2,4-D), and cytokinin such as kinetin, zeatin, and dihydrozeatin can beused.

[0143] As the vitamins, for example, biotin, thiamin (vitamin B₁),pyridoxine (vitamin B₆), pantothenic acid, inositol, nicotinic acid andthe like can be used.

[0144] As the amino acids, for example, glycine, phenylalanine, leucine,glutamine, cysteine and the like can be added.

[0145] Generally, the carbon source in a concentration of about 1-about30 g/l, the inorganic component in a concentration of about 0.1 μM-about100 mM, the phytohormones in a concentration of about 0.01-about 10 μM,and the vitamins and the amino acids respectively in a concentration ofabout 0.1-about 100 mg/l are used.

[0146] According to the second invention of the present application,both a liquid medium and such a solid medium that contains agar andgelan gum normally in an amount of 0.1-1% can be used. According to thetissue culture of the second invention of the present application, apiece of a tissue or a cell of a root, a growing point, a leaf, a stem,a seed, a pollen, an anther and a calyx and the like of the said plantor cultured cells which are obtained by the tissue culture thereof inthe above-mentioned medium or another conventional medium can be used.

[0147] The second invention of the present application can also beapplied to neoplastic cell and/or hairy-root, obtained by infection withAgrobacterium tumefaciens or Agrobacterium rhizogenes.

[0148] When these tissues or cells are cultured by controlling theoxygen concentration in the gas phase in the culture vessel to less thanthe oxygen concentration in the atmosphere, from the initial stage ofthe culture, or by controlling the dissolved oxygen concentration in thefluid medium which is in contact with the tissue or the cell to lessthan the saturated dissolved oxygen concentration at that temperature,from the initial stage of the culture, cultured tissue or the culturedcells having higher taxane-type diterpene productivity than that ofthose obtained by the tissue culture carried out under normal cultureconditions can be obtained.

[0149] According to the second invention of the present application, theinitial stage of the culture refers to from the time when the culturewas started through the 7th day after the start of the culture, and thecontrolling of the oxygen concentration in the gas phase in the culturevessel or the controlling of the dissolved oxygen concentration in thefluid medium which is in contact with the tissue or the cell ispreferably done from the beginning of the culture.

[0150] The controlling period is not particularly limited, and, thecontrolling under the said condition can be done in the entire cultureperiod, or only in a part of the entire culture period, however, it ispreferable to carry out the control at least for 3 days during theentire culture period.

[0151] The production method according to the second invention of thepresent application can be used together with a culture method carriedout in the presence of various kinds of taxane-type diterpene productionpromoting substances to further increase the productivity of thetaxane-type diterpene.

[0152] Examples of the taxane-type diterpene production promotingsubstance include, for example, jasmonic acids represented by theabove-mentioned general formulae (I), (II) or (III), compoundscontaining a heavy metal, complex ions containing a heavy metal, heavymetal ions, amines and antiethylene agents to be used for theabove-mentioned first invention of the present application.

[0153] Also the second invention of the present application can be alsoused together with the method of the third invention of the presentapplication which will be described later in detail, wherein the cellsare fractionated into a plurality of layers according to the differencein their specific gravities, and the cells contained in at least onelayer are cultured.

[0154] The production method according to the second invention of thepresent application can be used together with both the said methodaccording to the first invention of the present application whereinculture is carried out in the presence of the jasmonic acids and thelike and the method according to the third invention of the presentapplication wherein the cells are fractionated into a plurality oflayers according to the difference in their specific gravities and cellscontained in at least one layer are cultured.

[0155] Taxane-type diterpene can be fractionated from the cultures suchas cultured tissues, cultured cells and culture medium, which areobtained according to the above-mentioned process, by extraction with anorganic solvent such as methanol.

[0156] One preferable example of the tissue culture according to thesecond invention of the present application can be illustrated asfollows.

[0157] A piece of a plant body of a plant belonging to genus Taxus, suchas a root, a growing point, a leaf, a stem, a seed and the like issterilized and placed on Woody Plant Medium solidified with gelan gum,and kept at 10-35° C. for about 14-60 days so that a part of the tissuepiece is changed to callus. By subculturing the callus thus obtained,the growing speed is gradually increased and stabilized callus can beobtained. By the stabilized callus, we refer to a callus which remainsin callus state during culture without showing differentiation into ashoot or a root and the cells of which have uniform growing speed.

[0158] Such stabilized callus is transferred to a liquid medium, suitedfor the growth, such as liquid Woody Plant Medium and grown. The growingspeed is further increased in the liquid medium. According to thepresent invention, the stabilized callus or the cells constituting theabove-mentioned callus is grown under the culture conditions wherein theoxygen concentration in a gas phase in a culture vessel is controlled toless than the oxygen concentration in the atmosphere from the initialstage of the culture, or the dissolved oxygen concentration in a fluidmedium which is in contact with the tissue or the cell is controlled toless than the saturated dissolved oxygen concentration at thattemperature, from the initial stage of the culture.

[0159] The tissue or the cell gains energy necessary for maintenance andgrowth of individium, by consuming oxygen (respiration). It is generallyknown that when a tissue or a cell is cultured, the cell mass isincreased and the amount of the oxygen consumption is increased as wellwith the passage of culture period. Accordingly unless a ventilation gasis forcedly supplied from outside of the system, the oxygenconcentration in the gas phase in the culture vessel such as a flaskwherein the tissue or the cell is contained, or the dissolved oxygenconcentration in the medium which is in contact with the tissue or thecell naturally decreases to a value less than the oxygen concentrationin the atmosphere, or the saturated dissolved oxygen concentration atthat temperature, with the passage of culture period.

[0160] The present invention is different from the above-mentionedfinding on the point that the culture is carried out by activelycontrolling the oxygen concentration in the gas phase in the culturevessel which contains the tissue or the cell or the dissolved oxygenconcentration in the culture medium to less than the oxygenconcentration in the atmosphere or the saturated dissolved oxygenconcentration at that temperature.

[0161] In one illustrative process to enhance the effect of the presentinvention, the oxygen concentration in the gas phase in the culturevessel or the dissolved oxygen concentration in the fluid medium ispreviously controlled to less than the oxygen concentration in theatmosphere or the saturated dissolved oxygen concentration at thattemperature, prior to the subculture of the tissue or the cell in theculture vessel.

[0162] The controlling period is not particularly limited as mentionedabove, however, it is preferable to carry out the control at least for 3days during the entire culture period.

[0163] In addition to that, the controlling method is not particularlylimited to any as far as it is a method wherein the oxygen concentrationin the gas phase in the culture vessel which contains the tissue or thecell, or the dissolved oxygen concentration in the fluid medium which isin contact with the tissue or the cell, can be controlled to less thanthe oxygen concentration in the atmosphere or the saturated dissolvedoxygen concentration at that temperature, and in some examples of suchmethod, a gas having a controlled oxygen concentration, which isobtained by mixing an air with nitrogen and the like to lower the oxygenconcentration, is directly sent into the gas phase in the culture vesselor the culture medium, or such a gas is directly sent into the culturemedium outside of the culture vessel, i.e. in an aeration tank and thelike, then the culture medium is poured into the culture vessel, or agas such as air to be supplied to the culture vessel is directly sentinto the gas phase or the culture medium by controlling the feed speed,or such a gas is directly sent into the culture medium outside of theculture vessel, i.e. in an aeration tank and the like then the culturemedium is poured into the culture vessel, or the culture vessel isplaced under low oxygen atmosphere to carry out culture or the cultureis carried out in the presence of an oxygen adsorbing agent.

[0164] The temperature for the tissue culture according to the presentinvention is usually about 10-about 35° C., and preferably about 23-28°C. according to the high growing speed. As for the culture period, 14-42days are preferable.

[0165] When a liquid medium is used for the culture according to thepresent invention, the cultured cells can be fractionated from theculture medium after the culture is completed by such a method asdecantation or filtration and the desired taxane-type diterpene can befractionated from the cultured cells and/or the culture medium by such amethod as extraction with an organic solvent. It is also possible torecover the desired compound continuously during the culture by allowingan adsorbing agent or an appropriate organic solvent coexist in theculture system.

[0166] The third invention of the present application will be explainedas follows.

[0167] According to the third invention of the present application, alayer containing the cultured cells, which shall show high taxane-typediterpene productivity after being cultured, can be exemplified by alayer having the specific gravity of 1.07 or less.

[0168] In a generally known method to fractionate the cells according totheir specific gravities, a density gradient is formed by a medium forcentrifugal separation, and the cells are layered over it, thencentrifugal separation is carried out.

[0169] As a medium for centrifugal separation, Ficoll, Percoll (bothproduced by Pharmacia LKB Biotechnology Co. Ltd.,), sucrose and cesiumchloride and the like are used. In Examples, the density gradient wasproduced by the use of Ficoll, however, the medium is not particularlylimited to any substance as far as it does not damage the cells. Ficollhas been used for separation of cell granules and the like (Hess, R. etal., Nature 208 (1965), 856-858) or separation of animal cells (Walder,I. A. et al., Proc. Soc. exptl. Biol. Med., 112(1963) 494-496) and thelike.

[0170] The number of the layers forming the density gradient is notparticularly limited.

[0171] In Examples, a density gradient wherein the difference of thespecific gravity between each layer is 0.02 is formed by the layershaving the specific gravity of 1.03, 1.05, 1.07, 1.09 and 1.11 (g/ml),however, the difference of the specific gravity is not limited to thisvalue, and the difference of the specific gravity between each layer canbe the same or different.

[0172] Accordingly, the definition of the density gradient includes acase wherein the gradient changes continuously (the condition whereinthe number of the layers forming the density gradient is close toinfinite, and the difference of the specific gravity between each layeris close to 0).

[0173] The cells can be fractionated into a plurality of layersaccording to the difference in their specific gravities by thus formingthe density gradient, layering the cells and carrying out thecentrifugal separation.

[0174] The specific gravity of the layer to be formed is normally in therange of 1.00 to 1.20 g/ml, preferably in the range of 1.03 to 1.11g/ml. As a layer to become an object for culture, at least one layer isselected, but it is also possible to select all the layers and culturethem.

[0175] When a plurality of layers are selected and cells contained inthe selected layers are cultured, it is possible to culture the cells inthese layers individually, but, it is also possible to mix the cells intwo or more layers of the selected plurality of layers and culture them.

[0176] The cultured cells having high taxane-type diterpene productivitycan be usually obtained by culturing cells contained in a layer havingthe specific gravity of 1.07 or less, but, it is not always limited tothis range, since it may fluctuate depending on the cells to be culturedor the culture conditions. There is also a tendency that the cells in alayer of a higher specific gravity, have a higher content of thetaxane-type diterpene at the time when the fractionation is carried outaccording to the difference in the specific gravities. Accordingly, toensure that cultured cells which produce the taxane-type diterpene at ahigh rate can be obtained, it is desirable that the cells in all thefractionated layers are cultured for a certain period, then theconcentration of the taxane-type diterpene in the cells of each layer ismeasured, and the layer containing the cultured cells which produce thetaxane- type diterpene at a high rate is selected from among thoselayers.

[0177] So far, there have been no cases reported wherein the culturedcells of a plant producing the taxane-type diterpene are cultured afterthey are fractionated according to the specific gravity of the cells,and it was beyond all expectations that the cells can be fractionatedinto layers of cells each having different taxane-type diterpeneproductivity, by the difference of the specific gravities, and that thecells which produce the taxane-type diterpene at a high rate can beobtained by culture of cells which are contained in a layer having thespecific gravity of 1.07 or less, and whose taxane-type diterpenecontent is not so high at the time when they are fractionated.

[0178] According to the present invention, it is also possible tofractionate the cultured cells into a plurality of layers according tothe difference in the specific gravities by preparing a medium forcentrifugal separation having one particular specific gravity such as1.07 g/ml, for example, and carrying out the centrifugal separationaccording to the above-mentioned method.

[0179] The culture medium to be used for the present invention includestypical culture medium components. As such a component, an inorganiccomponent and a carbon source are typically used, and phytohormones,vitamins, and if necessary, amino acids can be added as well. As acarbon source, a disaccharide such as sucrose, maltose, and lactose,monosaccharide such as glucose, fructose and galactose, starch or amixture of two or more kinds of such sugar sources mixed at anappropriate ratio can be utilized.

[0180] As an inorganic component, illustrative examples includephosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron,manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine andcobalt, and these components can be added in the form of such a compoundas potassium nitrate, sodium nitrate, calcium nitrate, potassiumchloride, potassium monohydrogenphosphate, potassiumdihydrogenphosphate, calcium chloride, magnesium sulfate, sodiumsulfate,ferroussulfate, ferricsulfate, manganese sulfate, zinc sulfate, boricacid, copper sulfate, sodium molybdate, molybdenum trioxide, potassiumiodide, cobalt chloride and the like.

[0181] As the phytohormone, for example, auxin such as indoleaceacid(IAA), naphthalenacetic acid (NAA), 2,4-dichlorophenoxy acetic acid(2,4-D), and cytokinin such as kinetin, zeatin, dihydrozeatin can beused.

[0182] As the vitamins, for example, biotin, thiamin (vitamin B₁),pyridoxine(vitamin B₆), pantothenic acid, inositol, nicotinic acid andthe like can be used.

[0183] As the amino acids, for example, glycine, phenylalanine, leucine,glutamine, cysteine and the like can be added.

[0184] Generally, the inorganic component in a concentration of about0.1 μM-about 100 mM, the carbon source in a concentration of about1-about 30 g/l, the phytohormone in a concentration of about 0.01-about10 μM, and the vitamins and the amino acids respectively in aconcentration of about 0.1-about 100 mg/l are used.

[0185] Examples of a medium to be used for the present invention includethose known media which have been conventionally used for the planttissue culture, such as Medium of Murashige & Skoog (1962), medium ofLinsmaier Skoog (1965), Woody Plant Medium (1981), Gamborg's B-5 mediumand Mitsui's M-9 medium to which the above-mentioned phytohormone, andif necessary, the above-mentioned carbon source, vitamins and aminoacids are added.

[0186] According to the present invention, both a liquid medium and sucha solid medium that contains agar and gelan gum normally in an amount of0.1-1% can be used, however, usually a liquid medium is preferable.

[0187] According to the tissue culture of the present invention, a pieceof a tissue or a cell of a root, a growing point, a leaf, a stem, aseed, a pollen, an anther and a Calyx and the like of the said plant, orcultured cells which are obtained by the tissue culture thereof in thesaid medium or another conventional medium can be used.

[0188] By fractionating these cells into particular specific gravityranges then culturing them according to the present invention, culturedcells having higher taxane-type diterpene productivity, in comparisonwith those in the control area wherein no fractionation was carried out,can be obtained. The taxane-type diterpene can be fractionated fromthese cultured cells by extraction with an organic solvent such asmethanol.

[0189] One preferable example of the tissue culture according to thepresent invention can be illustrated as follows.

[0190] A piece of a plant body of a plant belonging to genus Taxus, suchas a root, a growing point, a leaf, a stem, a seed and the like issterilized and placed on Woody Plant Medium solidified with gelan gum,and kept at 10-35° C. for 14-60 days so that a part of the tissue pieceis changed to callus. By subculturing the callus thus obtained, thegrowing speed is gradually increased and stabilized callus can beobtained. By the stabilized callus, we refer to a callus which remainsin callus state during culture without showing differentiation into ashoot or a root and the cells of which have uniform growing speed.

[0191] Such stabilized callus is transferred to a liquid medium, suitedfor the growth, such as liquid Woody Plant Medium and grown. The growingspeed is further increased in the liquid medium.

[0192] The temperature for the tissue culture according to the presentinvention is usually about 10-about 35° C., and preferably about 23-28°C. according to the high growing speed. As for the culture period, 14-42days are preferable.

[0193] When a liquid medium is used for the culture according to thepresent invention, the cultured cells can be fractionated from theculture medium after the culture is completed, by such a method asdecantation or filtration and the desired taxane-type diterpene can befractionated from this by such a method as extraction with an organicsolvent.

[0194] According to the first invention and the second invention of thepresent application, taxane-type diterpene can be easily obtained inlarge quantity.

[0195] According to the third invention of the present application,cultured cells which produce taxane-type diterpene at a high rate can beobtained with a simple operation.

[0196] When the first, second or third invention of the presentapplication is to be industrially executed, the efficiency can befurther increased by employing the following fourth, fifth, sixth orseventh invention of the present application in an independent form orin a combined form.

[0197] That means, it is necessary to supply a gas containing oxygen toa culture liquid to culture tissues or cells of a plant which producestaxane-type diterpene. Normally, air is used for this purpose, however,after an intensive study, the present inventors found that thetaxane-type diterpene production can be efficiently carried out by theuse of a gas containing 0.03-10%, preferably 0.1-5% of carbon dioxide,as a gas to be introduced to a tank for culturing the tissues or thecells of the plant producing the taxane-type diterpene, and completedthe fourth invention of the present application.

[0198] The present inventors also found that the productivity of thetaxane-type diterpene in the cultures can be remarkably improved and thefluctuation of the taxane-type diterpene productivity due to thesubculture can be controlled by carrying out a two-stage culture of thetissue or the cell of the plant producing the taxane-type diterpene,comprising a first stage using a medium to which an oxidizing agent or awater soluble organic compound containing oxygen is added for obtainingthe tissues or the cells which is activated for production of thetaxane-type diterpene in the subsequent stage, and a second stage whichis carried out such conditions that promote the production of thetaxane-type diterpene, and completed the fifth invention of the presentapplication. Here, examples of the oxidizing agent includeperoxodisulfates such as potassium peroxodisulfate and hydrogenperoxide, and examples of the water soluble organic compound containingoxygen include dimethyl formamide, dimethyl sulfoxide, and ethyleneglycol and the like. The total concentration of the above-mentionedadditive in the culture medium is preferably 10⁻⁶M -10⁻¹M immediatelyafter the addition, and it is further preferable to control theconcentration to be in the range of 10⁻⁵M to 10⁻²M.

[0199] The present inventors also found that the high density culture ofthe tissue or the cell of the plant producing the taxane-type diterpenecan be carried out by inoculating the tissues or the cells in a culturemedium containing a saccharide in a concentration of 2-50 g/l,preferably 10-30 g/l, and/or nitrate ion in a concentration of 2-50mmol/l, preferably 10-30 mmol/l, then by adding a nutrient sourcesolution containing the saccharide in an amount of 0.2-5 g/l, preferably0.5-3 g/l, and/or nitrate ion in an amount of 0.2-5 mmol/l, preferably0.5-3 mmol/l per day with respect to the initial volume of the saidculture medium, continuously or intermittently to the culture medium,thereby the taxane-type diterpene production volume per culture vesselcan be remarkably increased and completed the sixth invention of thepresent application. Here, by the density, we refer to a cell mass pervolume of the culture solution in the culture vessel, which is shown interms of dry cell mass (g) per liter of the culture solution. Accordingto the sixth invention of the present application, it is preferable tocarry out culture while the culture medium is renewed by adding thenutrient source solution and simultaneously separating and removing thesame volume of the medium from the tissues or the cells and to recoverthe taxane-type diterpene from at least one selected from the resultingcultures, the medium recovered by removal during the culture, and themedium obtained at the end of the culture. The sixth invention of thepresent application is particularly effective in improving thetaxane-type diterpene productivity in the high density culture whereinthe density of the tissue or the cell of the above-mentioned plant atthe start of the culture with respect to the medium volume is 50 g freshweight/l or higher.

[0200] Furthermore, though the culture is normally finished when thecells of high density are obtained, the present inventors achieved,through the intensive study, the continuous culture by continuing theculture while the cells are removed, and after further examination,finally completed a continuous culture method, which is the seventhinvention of the present application. That means, the taxane-typediterpene can be produced with such a high rate that could be hardlyattained with the conventional process, by adding the fresh mediumcontinuously or intermittently in such a way that the specific renewingratio defined by the dimensionless number F=V_(I)/V/μ (wherein, V is thetotal volume of the culture medium in a culture tank, V_(I). is the feedspeed of the fresh medium, and μ is the specific growth rate of thetissues or the cells) is in the range of 0.1 to 10, and by recoveringthe taxane-type diterpene from the culture medium containing the tissuesor the cells which is continuously or intermittently taken out from thetank and/or the culture solution which does not contain the tissue northe cell and which is continuously or intermittently taken out from thetank, and completed the seventh invention of the present application. Itis further preferable to set the specific renewing ratio of the culturemedium, F, to 0.5-5. The saccharide concentration in the culturesolution is preferably 5-40 g/l, and the nitrate ion concentration inthe culture solution is preferably 10-40 mmol/l. The present inventioncan be effective with the cell density in terms of fresh cell weight perlitter of 50-500 g, however, the higher the density is as far as it isin a range wherein extremely vigorous stirring is not required, the moreefficiently the taxane-type diterpene can be produced, thus thepreferable density is 200 g or higher per liter.

[0201] In order to combine the above-mentioned fourth, fifth, sixth, orseventh invention of the present application with the above-mentionedthird invention of the present application, the cells obtained accordingto the third invention of the present application can be culturedaccording to the fourth, fifth, sixth or seventh invention of thepresent application to produce the desired taxane-type diterpene.

BRIEF DESCRIPTION OF THE DRAWINGS

[0202]FIG. 1. is a graph showing the change of the yield of the taxol inthe culture medium after adding 100 μM of methyl jasmonate.

[0203]FIG. 2 is a graph showing the change of the yield of the baccatinIII in the culture medium after adding 100 μM of methyl jasmonate.

[0204]FIG. 3 is a diagram illustrating an example of a culture apparatusused for carrying out the tissue culture according to the secondinvention of the present application. Each numeral used in the FIG. 3has the following meaning.

[0205]1 Air feed pipe

[0206]2 Nitrogen feed pipe

[0207]3 Culture vessel

[0208]4 Sparger for supplying oxygenic gas

[0209]5 Electrode for dissolved oxygen

[0210]6 Dissolved oxygen concentration controller

[0211]7 Vent

[0212]8 Valve

[0213]9 Oxygen flow control valve

[0214]10 Air filter

[0215]11 Impeller

[0216]FIG. 4 is a graph showing the growth in the culture after thefractionation.

[0217]FIG. 5 is a graph showing the taxane content in the culture afterthe fractionation.

[0218]FIG. 6 is a graph showing the distribution of the cells at thefractionation.

[0219]FIG. 7 is a graph showing the taxane content (in cell) at thefractionation.

[0220]FIG. 8 is a diagram illustrating an example of a culture apparatusused for carrying out the tissue culture according to the sixth or theseventh invention of the present application. Each numeral and alphabetused in the FIG. 8 has the following meaning.

[0221]12 Medium feed pipe

[0222]13 Medium feed opening

[0223]14 Opening with a filter for taking out culture medium alone (theculture medium containing no tissues nor cells)

[0224]15 Culture medium outlet pipe

[0225]16 Sparger for supplying oxygenic gas

[0226]17 Impeller

[0227]18 Culture mixture (the culture solution containing tissues orcells) discharge pipe

[0228]19 Pressurized fluid inlet a, b, c, d and e Valves

Best Mode for Carrying Out the Invention

[0229] The invention will be further illustrated with the followingexamples and comparative examples, however, these examples are not to beconstrued to limit the scope of the invention.

EXAMPLE 1

[0230] A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵M, and static culture was carried out at 25° C.in a dark place to provide callus of Taxus baccata LINN. One gram (freshweight) of the callus was inoculated to an Erlenmeyer flask containing20 ml of liquid Woody Plant Medium to which the above-mentionedcomponent was added to give the same concentration and shake culture wascarried out with a rotary shaker (amplification of 25 mm, 120 rpm) andthe callus was subcultured in every 21 days to accelerate the growthrate thereof.

[0231] One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component was added to give the sameconcentration, and shake culture was carried out at 25° C. for 14 days.On the 14th day after starting the culture, methyl ester of tuberonicacid (which is a compound represented by the general formula (I),wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵,and R^(6a) are hydrogen atoms, R^(6b) is hydroxyl group, R⁷ is methoxygroup, n is 1 and C³ and C⁴ have a double bond between them) was addedas the compound represented by the general formula (I) to give the finalconcentration of 0.01-1000 μM, and the culture was further carried outfor another 7 days.

[0232] After completing the culture, cultured cells of Taxus baccataLINN were harvested by filtration and lyophilized, then the dry weightwas measured to obtain the yield of the cultured cells per liter of theliquid medium. Taxane-type diterpenes were extracted from the driedcallus with methanol and the like, and they were determined by comparingwith standard taxol, cephalomannine, and baccatin III using highperformance liquid chromatography to measure the yields of thetaxane-type diterpenes. The results are shown in Table 1.

COMPARATIVE EXAMPLE 1

[0233] The procedure of Example 1 was carried out except that the methylester of tuberonic acid was not added. The results are shown in Table 1.

EXAMPLE 2

[0234] The procedure of Example 1 was carried out except that the methylester of tuberonic acid was added successively a total of 4 times inevery two days starting from the 7th day after starting the culture (togive the final concentration of 25 μM each time, and to give the totalconcentration of 100 μM). The results are shown in Table 1.

EXAMPLE 3

[0235] The procedure of Example 1 was carried out except that 100 μM ofthe methyl ester of tuberonic acid was added on the first day afterstarting the culture then the culture was further carried out foranother 20 days. The results are shown in Table 1.

EXAMPLE 4

[0236] The procedure of Example 1 was carried out except that 100 μM ofthe methyl ester of tuberonic acid was added on the 7th day afterstarting the culture and the culture was further carried out for another14 days. The results are shown in Table 1. TABLE 1 concentration yield*) yield *) of of yield *) of methyl cell baccatin of cephalo-tuberonate yield III taxol mannine (μM) (g/l) (mg/l) (mg/l) (mg/l)Comparative 0 14.3 0.4 3.5 1.2 Example 1 Example 1 0.01 14.3 0.4 4.6 1.3″ 0.1 13.5 0.5 5.5 1.5 ″ 1 13.2 0.7 6.9 1.8 ″ 10 13.0 0.9 14.8 2.2 ″ 10012.7 12.2 16.5 2.4 ″ 250 12.5 14.5 21.1 2.2 ″ 500 11.6 15.0 10.6 2.3 ″1000 7.5 1.2 3.9 1.8 Example 2 100 (25 × 4) 12.9 13.5 22.5 2.3 Example 3100 7.1 1.0 4.0 1.3 Example 4 100 12.5 10.5 15.6 2.1

EXAMPLE 5

[0237] The cells with the accelerated growth rate obtained by the methodof Example 1 were fractionated firstly by a stainless steel mesh andcell clusters having the size of 250-840 μm were obtained. A mediumhaving a specific gravity of 1.07 (g/ml) was produced by the use ofFicoll and the above-mentioned cells were layered over it andcentrifuged at 700 rpm for 6 minutes. The cells were fractionated intotwo layers according to the difference of the specific gravity. Thecells contained in the layer of 1.07 g/ml or less were fractionated andwashed with 2% sucrose solution three times or more, to wash off Ficoll.After the washing, 1 g (fresh weight) of the cells was transferred to anErlenmeyer flask containing 20 ml of liquid Woody Plant Medium and shakeculture was carried out at 25° C. for 14 days. On the 14th day afterstarting the culture, methyl ester of tuberonic acid was added to it togive the final concentration of 250 μM, and the culture was furthercarried out for another 7 days. After completing the culture, theprocedure of Example 1 was carried out. The results are shown in Table2. The productivity of the taxane-type diterpenes could be greatlyimproved by the combination of the selection of cells having aparticular specific gravity and the addition of the methyl ester oftuberonic acid.

COMPARATIVE EXAMPLE 2

[0238] The procedure of Example 5 was carried out except that the methylester of tuberonic acid was not added. The results are shown in Table 2.TABLE 2 concentration yield *) of yield *) yield *) of methyl cell of ofcephalo- tuberonate yield baccatin III taxol mannine (μM) (g/l) (mg/l)(mg/l) (mg/l) Comparative  0 14.2 0.5 5.4 1.5 Example 2 Example 5 25012.2 17.4 28.3 3.1

EXAMPLE 6

[0239] 250 μM of methyl ester of tuberonic acid was added to culturedcells of Taxus brevifolia NUTT obtained on the 14th day after startingthe culture by the method of Example 1, and the culture was furthercarried out for another 7 days. After completing the culture, theprocedure of Example 1 was carried out. The results are shown in Table3.

COMPARATIVE EXAMPLE 3

[0240] The procedure of Example 6 was carried out except that methylester of tuberonic acid was not added. The results are shown in Table 3.

EXAMPLE 7

[0241] The procedure of Example 6 was carried out except that culturedcells of T. media were used. The results are shown in Table 3.

COMPARATIVE EXAMPLE 4

[0242] The procedure of Example 7 was carried out except that the methylester of tuberonic acid was not added. The results are shown in Table 3.TABLE 3 concentration yield *) yield *) of of yield *) of methyl cellbaccatin of cephalo- tuberonate yield III taxol mannine (μM) (g/l)(mg/l) (mg/l) (mg/l) Comparative  0 12.5 0.1 0.2 0.2 Example 3 Example 6250 10.0 1.4 3.3 0.5 Comparative  0 13.6 0.2 0.3 0.1 Example 4 Example 7250 9.5 10.4 5.4 0.5

EXAMPLE 8

[0243] A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵M, and static culture was carried out at 25° C.in a dark place to provide callus of Taxus baccata LINN. One gram (freshweight) of the callus was inoculated to an Erlenmeyer flask containing20 ml of liquid Woody Plant Medium to which the above-mentionedcomponent was added to give the same concentration and shake culture wascarried out with a rotary shaker (amplification of 25 mm, 120 rpm) andthe callus was subcultured in every 21 days to accelerate the growthrate thereof.

[0244] One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component was added to give the sameconcentration, and shake culture was carried out at 25° C. for 14 days.On the 14th day after starting the culture, methyl ester of cucurbicacid (which is a compound represented by the general formula (II)wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³ R⁴, R⁵,and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1 and C³ and C⁴have a double bond between them) was added as one of jasmonic acids togive the final concentration of 0.01-1000 μM, and the culture wasfurther carried out for another 7 days.

[0245] After completing the culture, cultured cells of Taxus baccataLINN were harvested by filtration and lyophilized, then the dry weightwas measured to obtain the yield of the cultured cells per liter of theliquid medium. Taxane-type diterpenes were extracted from the driedcallus with methanol and the like, and they were determined by comparingwith standard taxol, cephalomannine, and baccatin III using highperformance liquid chromatography to measure the yields of thetaxane-type diterpenes. The results are shown in Table 4.

COMPARATIVE EXAMPLE 5

[0246] The procedure of Example 8 was carried out, except that themethyl ester of cucurbic acid was not added. The results are shown inTable 4.

EXAMPLE 9

[0247] The procedure of Example 8 was carried out except that the methylester of cucurbic acid was added successively a total of 4 times inevery two days starting from the 7th day after starting the culture (togive the final concentration of 25 μM each time, and to give the totalconcentration of 100 μM). The results are shown in Table 4.

EXAMPLE 10

[0248] The procedure of Example 8 was carried out except that 100 μM ofthe methyl ester of cucurbic acid was added on the first day afterstarting the culture then the culture was further carried out foranother 20 days. The results are shown in Table 4.

EXAMPLE 11

[0249] The procedure of Example 8 was carried out except that 100 μM ofthe methyl ester of cucurbic acid was added on the 7th day afterstarting the culture and the culture was further carried out for another14 days. The results are shown in Table 4. TABLE 4 concentration yield*) yield *) of of yield *) of methyl cell baccatin of cephalo- cucurbateyield III taxol mannine (μM) (g/l) (mg/l) (mg/l) (mg/l) Comparative 011.3 0.2 3.3 2.4 Example 5 Example 8 0.01 11.3 0.3 4.3 2.6 ″ 0.1 11.10.3 5.4 3.1 ″ 1 10.4 0.3 6.7 3.3 ″ 10 10.4 0.5 11.3 4.0 ″ 100 9.8 7.215.7 4.4 ″ 250 9.6 10.6 17.7 5.3 ″ 500 9.3 12.0 13.1 3.0 ″ 1000 5.7 1.24.2 2.8 Example 9 100 (25 × 4) 10.5 14.6 18.2 4.1 Example 10 100 6.1 0.83.9 2.8 Example 11 100 9.5 8.4 16.8 4.1

EXAMPLE 12

[0250] the cells with the accelerated growth rates obtained by themethod of Example 8 were fractionated firstly by a stainless steel meshand cell clusters having the size of 250-840 μm were obtained. A mediumhaving a specific gravity of 1.07 (g/ml) was produced by the use ofFicoll and the above-mentioned cells were layered over it andcentrifuged at 700 rpm for 6 minutes. The cells were fractionated intotwo layers according to the difference of the specific gravity. Thecells contained in the layer of 1.07 g/ml or less were fractionated andwashed with 2% sucrose solution three times or more, to wash off Ficoll.After the washing, 1 g (fresh weight) of the cells was inoculated to anErlenmeyer flask containing 20 ml of liquid Woody Plant Medium and shakeculture was carried out at 25° C. for 14 days. On the 14th day afterstarting the culture, methyl ester of cucurbic acid was added to it togive the final concentration of 250 μM, and culture was further carriedout for another 7 days. After completing the culture, the procedure ofExample 8 was carried out. The results are shown in Table 5. Theproductivity of the taxane-type diterpenes could be greatly improved bythe combination of the selection of cells having a particular specificgravity and the addition of the methyl ester of cucurbic acid.

COMPARATIVE EXAMPLE 6

[0251] The procedure of Example 12 was carried out except that themethyl ester of cucurbic acid was not added. The results are shown inTable 5. TABLE 5 concentration yield *) of yield *) yield *) of methylcell of of cephalo- cucurbate yield baccatin III taxol mannine (μM)(g/l) (mg/l) (mg/l) (mg/l) Comparative  0 11.5 0.3 4.3 2.8 Example 6Example 12 250 9.7 22.2 29.7 3.8

EXAMPLE 13

[0252] 250 μM of methyl ester of cucurbic acid was added to culturedcells of Taxus brevifolia NUTT obtained on the 14th day after startingthe culture by the method of Example 8, and the culture was furthercarried out for another 7 days. After completing the culture, theprocedure of Example 8 was carried out. The results are shown in Table6.

COMPARATIVE EXAMPLE 7

[0253] The procedure of Example 13 was carried out except that methylester of cucurbic acid was not added. The results are shown in Table 6.

EXAMPLE 14

[0254] The procedure of Example 13 was carried out except that culturedcells of T. media were used. The results are shown in Table 6.

COMPARATIVE EXAMPLE 8

[0255] The procedure of Example 14 was carried out except that themethyl ester of cucurbic acid was not added. The results are shown inTable 6. TABLE 6 concentration yield *) yield *) of of yield *) ofmethyl cell baccatin of cephalo- cucurbate yield III taxol mannine (μM)(g/l) (mg/l) (mg/l) (mg/l) Comparative  0 12.5 0.1 0.2 0.2 Example 7Example 13 250 11.0 1.2 1.3 0.3 Comparative  0 13.6 0.2 0.3 0.1 Example8 Example 14 250 12.3 5.2 3.9 0.2

EXAMPLE 15

[0256] A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵M, and static culture was carried out at 25° C.in a dark place to provide callus of Taxus baccata LINN. One gram (freshweight) of the callus was inoculated to an Erlenmeyer flask containing20 ml of liquid Woody Plant Medium to which the above-mentionedcomponent was added to give the same concentration and shake culture wascarried out with a rotary shaker (amplification of 25 mm, 120 rpm) andthe callus was subcultured in every 21 days to accelerate the growthrate thereof.

[0257] One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component was added to give the sameconcentration, and shake culture was carried out at 25° C. for 14 days.On the 14th day after starting the culture, methyl ester of jasmonicacid (which is a compound represented by the general formula (III)wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵,and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1 and C³ and C⁴have a double bond between them, 90% of which is in the trans-form and10% of which is in the cis-form) was added as one of jasmonic acids togive the final concentration of 0.01-1000 μM, and the culture wasfurther carried out for another 7 days.

[0258] After completing the culture, cultured cells of Taxus baccataLINN were harvested by filtration and lyophilized, then the dry weightwas measured to obtain the yield of the cultured cells per liter of theliquid medium. Taxane-type diterpenes were extracted from the driedcallus with methanol and the like, and they were determined by comparingwith standard taxol, cephalomannine, and baccatin III using highperformance liquid chromatography to measure the yields of thetaxane-type diterpenes. The results are shown in Table 7.

COMPARATIVE EXAMPLE 9

[0259] The procedure of Example 15 was carried out except that themethyl ester of jasmonic acid was not added. The results are shown inTable 7.

EXAMPLE 16

[0260] The procedure of Example 15 was carried out except that themethyl ester of jasmonic acid was added successively a total of 4 timesin every two days starting from the 7th day after starting the culture(to give the final concentration of 25 μM each time, and to give thetotal concentration of 100 μM). The results are shown in Table 7.

EXAMPLE 17

[0261] The procedure of Example 15 was carried out except that 100 μM ofthe methyl ester of jasmonic acid was added on the first day afterstarting the culture then the culture was further carried out foranother 20 days. The results are shown in Table 7.

EXAMPLE 18

[0262] The procedure of Example 15 was carried out except that 100 μM ofthe methyl ester of jasmonic acid was added on the 7th day afterstarting the culture and the culture was further carried out for another14 days. The results are shown in Table 7.

EXAMPLE 19

[0263] The procedure of Example 15 was carried out except that jasmonicacid (which is a compound represented by the general formula (III)wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵,and R⁶ are hydrogen atoms, R⁷ is hydroxy group, n is 1 and C³ and C⁴have a double bond between them; 90% of which is in the trans- form and10% of which is in the cis-form) was added as one of jasmonic acids togive the final concentration of 0.01-1000 μM. The results are shown inTable 8.

COMPARATIVE EXAMPLE 10

[0264] The procedure of Example 19 was carried out except that themethyl ester of jasmonic acid was not added. The results are shown inTable 8.

EXAMPLE 20

[0265] The analytical results of taxane-type diterpenes existed in theculture medium of Example 15 prior to the addition of 100 μM of methyljasmonate, on the third day after the addition, and on the 7th day afterthe addition, are shown in FIG. 1 and FIG. 2 On the 7th day of theculture, about half of the taxol and about 70% of the baccatin III wereleaked in the medium.

COMPARATIVE EXAMPLE 11

[0266] The procedure of Example 20 was carried out except that themethyl ester of jasmonic acid was not added. The results are shown inFIG. 1 and FIG. 2.

EXAMPLE 21

[0267] The cells with accelerated growth rate obtained by the method ofExample 15 were fractionated firstly by a stainless steel mesh and cellclusters having the size of 250-840 μm were obtained. A medium having aspecific gravity of 1.07 (g/ml) was produced by the use of Ficoll andthe above-mentioned cells were layered over it and centrifuged at 700rpm for 6 minutes. The cells were fractionated into two layers accordingto the difference of the specific gravity. The cells contained in thelayer of 1.07 g/ml or less were fractionated and washed with 2% sucrosesolution three times or more, to wash off Ficoll. After the washing, 1 g(fresh weight) of the cells was inoculated to an Erlenmeyer flaskcontaining 20 ml of liquid Woody Plant Medium and shake culture wascarried out at 25° C. for 14 days. On the 14th day after starting theculture, methyl ester of jasmonic acid was added to it to give the finalconcentration of 250 μM, and the culture was further carried out foranother 7 days. After completing the culture, the procedure of Example15 was carried out. The results are shown in Table 9. The productivityof the taxane-type diterpenes could be greatly improved by thecombination of the selection of cells having a particular specificgravity and the addition of the methyl ester of jasmonic acid.

COMPARATIVE EXAMPLE 12

[0268] The procedure of Example 21 was carried out except that themethyl ester of jasmonic acid was not added. The results are shown inTable 9.

EXAMPLE 22

[0269] 250 μM of methyl ester of jasmonic acid was added to culturedcells of Taxus brevifolia NUTT obtained on the 14th day after startingthe culture by the method of Example 15, and the culture was furthercarried out for another 7 days. After completing the culture, theprocedure of Example 15 was carried out. The results are shown in Table10.

COMPARATIVE EXAMPLE 13

[0270] The procedure of example 22 was carried out except that methylester of jasmonic acid was not added. The results are shown in Table 10

EXAMPLE 23

[0271] the procedure of example 22 was carried out except that culturedcells of T. media were used. The results are shown in Table 10.

COMPARATIVE EXAMPLE 14

[0272] The procedure of example 23 was carried out except that themethyl ester of jasmonic acid was not added. The results are shown inTable 10. TABLE 7 concentration yield *) yield *) of of yield *) ofmethyl cell baccatin of cephalo- jasmonate yield III taxol mannine (μM)(g/l) (mg/l) (mg/l) (mg/l) Comparative 0 12.2 0.2 2.8 1.5 Example 9Example 15 0.01 12.2 0.3 3.2 1.6 ″ 0.1 12.1 0.3 4.2 1.6 ″ 1 11.3 0.4 4.91.8 ″ 10 11.3 0.7 10.8 2.1 ″ 50 10.9 3.1 10.9 2.2 ″ 100 9.8 9.2 13.5 2.0″ 250 10.0 12.9 15.1 2.0 ″ 500 10.6 13.0 12.6 1.9 ″ 1000 5.9 1.2 3.1 1.8Example 16 100 (25 × 4) 10.7 14.9 16.7 2.2 Example 17 100 7.2 1.2 4.51.7 Example 18 100 9.7 10.6 14.2 2.1

[0273] TABLE 8 concentration yield *) yield *) of of yield *) ofjasmonic cell baccatin of cephalo- acid yield III taxol mannine (μM)(g/l) (mg/l) (mg/l) (mg/l) Comparative 0 12.2 0.2 1.2 0.4 Example 10Example 19 0.01 12.2 0.3 2.2 0.6 ″ 0.1 12.2 0.3 3.4 0.6 ″ 1 11.6 0.3 5.90.8 ″ 10 11.5 0.5 8.3 2.6 ″ 50 11.4 2.5 10.2 2.2 ″ 100 10.3 7.2 12.8 3.0″ 250 10.1 10.6 14.7 2.7 ″ 500 10.2 12.0 11.1 1.1 ″ 1000 6.7 1.2 1.2 0.5

[0274] TABLE 9 concentration yield *) yield *) of of yield *) of methylcell baccatin of cephalo- jasmonate yield III taxol mannine (μM) (g/l)(mg/l) (mg/l) (mg/l) Comparative  0 12.6 0.5 6.3 2.2 Example 12 Example21 250 10.2 18.7 43.1 3.2

[0275] TABLE 10 concentration yield *) yield *) of of yield *) of methylcell baccatin of cephalo- jasmonate yield III taxol mannine (μM) (g/l)(mg/l) (mg/l) (mg/l) Comparative  0 12.5 0.1 0.2 0.2 Example 13 Example22 250 10.2 3.4 4.3 0.5 Comparative  0 14.2 0.2 0.3 0.1 Example 14Example 23 250 12.6 12.4  4.4 0.2

EXAMPLE 24

[0276] A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵M, and static culture was carried out at 25° C.in a dark place to provide callus of Taxus baccata LINN. One gram (freshweight) of the callus was inoculated to an Erlenmeyer flask containing20 ml of liquid Woody Plant Medium to which the above-mentionedcomponent was added to give the same concentration and shake culture wascarried out with a rotary shaker (amplification of 25 mm, 100 rpm) andthe callus was subcultured in every 21 days to accelerate the growthrate thereof.

[0277] One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component was added to give the sameconcentration, and [Ag(S₂O₃)₂]³⁻ was added to it as a compoundcontaining a heavy metal to give the final concentration of 10⁻⁹M-1M.Then shake culture was carried out at 25° C. for 21 days.

[0278] After completing the culture, cultured cells of Taxus baccataLINN were harvested by filtration and lyophilized, then the dry weightwas measured to obtain the growth rate thereof. Taxane-type diterpeneswere extracted from the dried callus with methanol and the like, andthey were determined by comparing with standard taxol, cephalomannine,and baccatin III using high performance liquid chromatography to measurethe yields of the taxane-type diterpenes. The results are shown in Table11.

EXAMPLE 25

[0279] The procedure of Example 24 was carried out except that[Ag(S₂O₃)₂]³⁻ was added on the 7th day after starting the culture togive the final concentration of 10⁻³M and the culture was furthercarried out for another 14 days. After the completion of the culture,the procedure of the Example 24 was carried out. The results are shownin Table 11.

EXAMPLE 26

[0280] The procedure of Example 24 was carried out except that[Ag(S₂O₃)₂]³⁻ was added on the 14th day after starting the culture togive the final concentration of 10⁻³M and the culture was furthercarried out for another 7 days. After the completion of the culture, theprocedure of the Example 24 was carried out. The results are shown inTable 11.

EXAMPLE 27

[0281] The procedure of Example 24 was carried out, except that[Ag(S₂O₃)₂]³⁻ was added on the 18th day after starting the culture togive the final concentration of 10⁻³M and the culture was furthercarried out for another 3 days. After the completion of the culture, theprocedure of Example 24 was carried out. The results are shown in Table11.

EXAMPLE 28

[0282] The procedure of Example 24 was carried out, except that[Ag(S₂O₃)₂]³⁻ was added successively a total of 5 times at 4 days'intervals starting from the start (0 day) of the culture (to give thefinal concentration of 2×10⁻⁴M each time, and to give the totalconcentration of 10⁻³M) The results are shown in Table 11.

EXAMPLE 29

[0283] The procedure of Example 24 was carried out except that methylester of jasmonic acid (which is a compound represented by the generalformula (III) wherein R^(1a), R^(1b), R^(1c), Rd, R^(1e), R^(1f), R²,R³, R⁴, R⁵, and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1 andC³ and C⁴ have a double bond between them) was added on the 14th dayafter starting the culture to give the final concentration of 10⁻⁴M. Theresults are shown in Table 11

EXAMPLE 30

[0284] The procedure of Example 24 was carried out except that the flaskwas put in a vessel (the capacity of 3000 ml) having a gas feed openingand a gas discharge opening; then the vessel was closed hermetically,and air was so mixed with nitrogen that the concentration of the oxygenin a gas to be supplied to the cells to be cultured became 10%, and thegas was supplied through the feed opening at the rate of 25 ml perminute. The results are shown in Table 11.

EXAMPLE 31

[0285] The procedure of Example 30 was carried out except that methylester of jasmonic acid was added on the 14th day of the culture to givethe final concentration of 10⁻⁴M. The results are shown in Table 11.

EXAMPLE 32

[0286] The procedure of Example 24 was carried out except that silvernitrate Ag(NO₃) of 10⁻³M was added at the start (0 day) of the cultureinstead of [Ag(S₂O₃)₂]³⁻. The results are shown in Table 11.

EXAMPLE 33

[0287] The procedure of Example 32 was carried out except that silvernitrate of 10⁻³M was added on the 14th day of the culture. The resultsare shown in Table 11.

COMPARATIVE EXAMPLE 15

[0288] The procedure of Example 24 was carried out except that[Ag(S₂O₃)₂]³⁻ was not added. The results are shown in Table 11.

EXAMPLE 34

[0289] The procedure of Example 24 was carried out except that cobaltchloride (CoCl₂) was added instead of [Ag(S₂O₃)₂]³⁻ as a compoundcontaining the heavy metal to give the final concentration of 10⁻⁹M-1M.The results are shown in Table 12.

EXAMPLE 35

[0290] The procedure of Example 34 was carried out except that cobaltchloride (CoCl₂) was added instead of [Ag(S₂O₃)₂]³⁻ to give the finalconcentration of 10⁻⁵M on the 7th day after starting the culture and theculture was further carried out for another 14 days. After completingthe culture, the procedure of Example 34 was carried out. The resultsare shown in Table 12.

EXAMPLE 36

[0291] The procedure of Example 34 was carried out except that cobaltchloride was added to give the final concentration of 10⁻⁵M on the 14thday after starting the culture and the culture was further carried outfor another 7 days. After completing the culture, the process of Example34 was carried out. The results are shown in Table 12.

EXAMPLE 37

[0292] The procedure of Example 34 was carried out except that cobaltchloride was added to give the final concentration of 10⁻⁵M on the 18thday after starting the culture, and the culture was further carried outfor another 3 days. After completing the culture, the procedure ofExample 34 was carried out. The results are shown in Table 12.

EXAMPLE 38

[0293] The procedure of Example 34 was carried out except that cobaltchloride was added successively a total of 5 times at 4 days' intervalsstarting from the start (0 day) of the culture (to give the finalconcentration of 2×10⁻⁶M each time, and to give the total concentrationof 10⁻⁵M). The results are shown in Table 12.

EXAMPLE 39

[0294] The procedure of Example 34 was carried out except that methylester of jasmonic acid (which is a compound represented by the generalformula (III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f),R², R³, R⁴, R⁵, and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1and C³ and C⁴ have a double bond between them) was added as one ofjasmonic acids to give the final concentration of 10⁻⁴M on the 14th dayafter starting the culture. The results are shown in Table 12.

EXAMPLE 40

[0295] The procedure of Example 34 was carried out except that the flaskwas put in a vessel (the capacity of 3000 ml) having a gas feed openingand a gas discharge opening, then the vessel was closed hermetically,and air was so mixed with nitrogen that the concentration of the oxygenin a gas to be supplied to the cells to be cultured became 10%, and thegas was supplied through the feed opening at the rate of 25 ml perminute. The results are shown in Table 12.

EXAMPLE 41

[0296] The procedure of Example 40 was carried out except that methylester of jasmonic acid was added to give the final concentration of10⁻⁴M on the 14th day of the culture. The results are shown in Table 12.TABLE 11 concentration concentration concentration max oxygen yield^(a))yield^(a)) of yield^(a)) total yield^(b)) of silver of silver of methylconcentration growth of cephalo- of of thiosulfare nitrate jasmonate inthe gas rate baccatin mannine taxol taxane (M) (M) (M) phase (%) (times)III (mg/l) (mg/l) (mg/l) (mg/l) Comparative 0 0 0 20 2.7 0.14 0.56 0.280.98 Example 15 Example 24 10⁻⁹ 0 0 20 2.7 0.20 0.56 0.33 1.09 ″ 10⁻⁸ 00 20 2.8 0.29 0.97 0.76 2.02 ″ 10⁻⁷ 0 0 20 2.7 0.34 2.47 1.57 4.38 ″10⁻⁶ 0 0 20 2.6 1.06 2.86 1.80 5.72 ″ 10⁻⁵ 0 0 20 2.7 1.13 4.10 7.9513.18 ″ 10⁻⁴ 0 0 20 2.7 7.62 5.11 14.53 27.26 ″ 10⁻³ 0 0 20 2.7 27.905.96 25.13 58.99 ″ 10⁻² 0 0 20 2.3 0.72 5.50 5.80 12.02 ″ 10⁻¹ 0 0 201.9 0.26 2.15 1.37 3.78 ″ 1 0 0 20 1.2 0.08 0.36 0.20 0.64 Example 2510⁻³ 0 0 20 2.2 11.71 4.86 10.64 27.21 Example 26 10⁻³ 0 0 20 2.5 2.293.83 4.47 10.59 Example 27 10⁻³ 0 0 20 2.9 0.28 1.44 1.55 3.27 Example28 10⁻³ 0 0 20 2.4 4.15 7.27 20.94 32.36 Example 29 10⁻³ 0 10⁻⁴ 20 2.725.14 12.57 33.61 71.32 Example 30 10⁻³ 0 0 10 2.7 22.81 9.08 27.4559.34 Example 31 10⁻³ 0 10⁻⁴ 10 2.7 27.00 12.34 34.73 74.07 Example 32 010⁻³ 0 20 2.8 3.01 7.54 12.66 23.21 Example 33 0 10⁻³ 0 20 2.8 2.18 6.4911.91 20.58

[0297] TABLE 12 concentration concentration max oxygen yield^(a))yield^(a)) of yield^(a)) total yield^(b)) of cobalt of methylconcentration growth of cephalo- of of chloride jasmonate in the gasrate baccatin mannine taxol taxane (M) (M) phase (%) (times) III (mg/l)(mg/l) (mg/l) (mg/l) Comparative 0 0 20 2.7 0.14 0.56 0.28 0.98 Example15 Example 34 10⁻⁹ 0 20 2.5 0.15 0.56 0.27 0.98 ″ 10⁻⁸ 0 20 2.6 0.150.61 0.29 1.05 ″ 10⁻⁷ 0 20 2.6 0.16 0.63 0.29 1.08 ″ 10⁻⁶ 0 20 2.4 2.255.79 8.32 16.36 ″ 10⁻⁵ 0 20 3.2 5.68 6.50 16.11 28.29 ″ 10⁻⁴ 0 20 2.71.64 3.46 4.92 10.02 ″ 10⁻³ 0 20 2.2 1.57 2.03 4.38 7.98 ″ 10⁻² 0 20 1.40.44 0.78 0.92 2.14 ″ 10⁻¹ 0 20 0.8 0.11 0.42 0.55 1.08 ″ 1 0 20 0.6 0 00 0 Example 35 10⁻⁵ 0 20 3.2 4.36 5.98 10.22 20.56 Example 36 10⁻⁵ 0 203.1 2.01 4.37 7.10 13.48 Example 37 10⁻⁵ 0 20 2.9 1.17 1.11 1.32 3.60Example 38 10⁻⁵ 0 20 2.6 5.32 6.58 15.76 27.66 Example 39 10⁻⁵ 10⁻⁴ 202.5 23.00 9.46 30.32 62.78 Example 40 10⁻⁵ 0 10 2.7 17.61 7.99 26.6452.24 Example 41 10⁻⁵ 10⁻⁴ 10 2.4 25.85 15.21 33.77 74.83

EXAMPLE 42

[0298] A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵M, and static culture was carried out at 25° C.in a dark place to provide Taxus baccata LINN callus. One gram (freshweight) of the callus was inoculated to an Erlenmeyer flask containing20 ml of liquid Woody Plant Medium to which the above-mentionedcomponent was added to give the same concentration and shake culture wascarried out with a rotary shaker (amplification of 25 mm, 100 rpm) andthe callus was subcultured in every 21 days to accelerate the growthrate thereof.

[0299] One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component had been added to give thesame concentration, then spermidine was added to it as amine to give thefinal concentration of 10⁻⁹M-1M. Then shake culture was carried out at25° C. for 21 days.

[0300] After completing the culture, cultured cells of Taxus baccataLINN were harvested by filtration and lyophilized, then the dry weightwas measured to obtain the growth rate thereof. Taxane-type diterpeneswere extracted from the dried cells with methanol and the like, and theywere determined by comparing with standard taxol, cephalomannine, andbaccatin III using high performance liquid chromatography to measure theyields of the taxane-type diterpene. The results are shown in Table 13.

EXAMPLE 43

[0301] The procedure of Example 42 was carried out except thatspermidine was added to give the final concentration of 10⁻⁵M on the 7thday after starting the culture and the culture was further continued foranother 14 days. After completing the culture, the procedure of Example42 was carried out. The results are shown in Table 13.

EXAMPLE 44

[0302] The procedure of Example 42 was carried out except thatspermidine was added to give the final concentration of 10⁻⁵M on the14th day after starting the culture and the culture was furthercontinued for another 7 days. After completing the culture, theprocedure of Example 42 was carried out. The results are shown in Table13.

EXAMPLE 45

[0303] The procedure of Example 42 was carried out except thatspermidine was added to give the final concentration of 10⁻⁵M on the18th day after starting the culture and the culture was furthercontinued for another 3 days. After completing the culture, theprocedure of Example 42 was carried out. The results are shown in Table13.

EXAMPLE 46

[0304] The procedure of Example 42 was carried out except thatspermidine was added successively a total of 5 times at 4 days intervalsstarting from the start (0 day) of the culture (to give the finalconcentration of 2×10⁻⁶M each time, and to give the total concentrationof 10⁻⁵M). The results are shown in Table 13.

EXAMPLE 47

[0305] The procedure of Example 42 was carried out except that methylester of jasmonic acid (which is a compound represented by the generalformula (III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f),R², R³, R⁴, R⁵, and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1and C³ and C⁴ have a double bond between them) was added as one ofjasmonic acids on the 14th day after starting the culture to give thefinal concentration of 10⁻⁴M. The results are shown in Table 13.

EXAMPLE 48

[0306] The procedure of Example 42 was carried out except that the flaskwas placed in a chamber (the capacity of 3000 ml) having a gas feedopening and a gas discharge opening, then the chamber was closedhermetically, and air was so mixed with nitrogen that the concentrationof the oxygen in a gas to be supplied to the cells to be cultured became10% and the gas was supplied through the feed opening at the rate of 25ml per minute. The results are shown in Table 13.

EXAMPLE 49

[0307] The procedure of Example 48 was carried out except that methylester of jasmonic acid was added to give the final concentration of10⁻⁴M on the 14th day of the culture. The results are shown in Table 13.

COMPARATIVE EXAMPLE 16

[0308] The procedure of Example 42 was carried out except thatspermidine was not added. The results are shown in Tables 13-15.

EXAMPLE 50

[0309] The procedure of Example 42 was carried out except that sperminwas added instead of spermidine to give the final concentration of10⁻⁹M-1M. The results are shown in Table 14.

EXAMPLE 51

[0310] The procedure of Example 42 was carried out except thatputrescine was added instead of spermin to give the final concentrationof 10⁻⁹M-1M. The results are shown in Table 15. TABLE 13 concentrationconcentration max oxygen yield^(a)) yield^(a)) of yield a) totalyield^(b)) of of methyl concentration growth of cephalo- of ofspermidine jasmonate in the gas rate baccatin mannine taxol taxane (M)(M) phase (%) (times) III (mg/l) (mg/l) (mg/l) (mg/l) Comparative 0 0 202.7 0.14 0.56 0.28 0.98 Example 16 Example 42 10⁻⁹ 0 20 2.9 0.31 0.580.32 1.21 ″ 10⁻⁸ 0 20 3.1 0.66 1.74 1.99 4.39 ″ 10⁻⁷ 0 20 3.1 1.34 5.7911.76 18.89 ″ 10⁻⁶ 0 20 3.2 3.61 6.04 13.51 23.16 ″ 10⁻⁵ 0 20 3.1 6.597.30 19.28 33.17 ″ 10⁻⁴ 0 20 3.1 2.78 6.49 12.96 22.23 ″ 10⁻³ 0 20 2.92.63 4.59 10.09 17.31 ″ 10⁻² 0 20 2.0 0.82 2.42 3.89 7.13 ″ 10⁻¹ 0 202.1 0.41 1.00 1.94 3.35 ″ 1 0 20 1.4 0.10 0.03 0.08 0.21 Example 43 10⁻⁵0 20 2.9 6.08 6.13 15.24 27.45 Example 44 10⁻⁵ 0 20 2.9 5.46 5.32 10.2020.98 Example 45 10⁻⁵ 0 20 3.0 2.95 2.90 5.63 11.48 Example 46 10⁻⁵ 0 202.9 6.30 6.01 15.43 27.74 Example 47 10⁻⁵ 10⁻⁴ 20 2.6 18.96 9.74 21.5550.25 Example 48 10⁻⁵ 0 10 2.7 13.43 7.81 19.05 40.29 Example 49 10⁻⁵10⁻⁴ 10 2.6 19.65 8.57 29.67 57.89

[0311] TABLE 14 concen- yield ^(a)) yield ^(a)) total tration of ofyield ^(a)) yield ^(b)) of growth baccatin cephalo- of of spermin rateIII mannine taxol taxane (M) (times) (mg/l) (mg/l) (mg/l) (mg/l)Comparative  0 2.7 0.14 0.56 0.28 0.98 Example 16 Example 50 10⁻⁹ 2.90.50 0.64 0.72 1.86 ″ 10⁻⁸ 2.3 0.62 1.01 1.79 3.42 ″ 10⁻⁷ 2.4 0.68 1.322.17 4.17 ″ 10⁻⁶ 2.4 0.89 1.97 4.03 6.89 ″ 10⁻⁵ 2.1 2.09 5.63 9.46 17.18″ 10⁻⁴ 2.3 1.64 4.09 6.23 11.96 ″ 10⁻³ 2.0 0.76 2.68 4.78 8.22 ″ 10⁻²2.1 0.70 1.54 1.06 3.30 ″ 10⁻¹ 1.4 0.39 0.32 0.58 1.29 ″  1 1.0 0.100.04 0.08 0.22

[0312] TABLE 15 yield^(a)) yield^(a)) total concentra- of of ceph-yield^(a)) yield^(b)) tion of growth baccatin aloman- of of putrescinerate III nine taxol taxane (M) (times) (mg/l) (mg/l) (mg/l) (mg/l)Comparative 0  2.7 0.14 0.56 0.28 0.98 Example 16 Example 51 10⁻⁹ 2.80.36 0.70 0.69 1.75 ″ 10⁻⁸ 2.8 0.40 1.02 1.52 2.94 ″ 10⁻⁷ 2.7 1.10 2.313.47 6.88 ″ 10⁻⁶ 3.0 5.74 5.51 8.19 19.44 ″ 10⁻⁵ 3.1 6.98 5.37 11.4423.79 ″ 10⁻⁴ 2.6 6.60 5.25 10.80 22.65 ″ 10⁻³ 2.2 1.95 1.88 3.07 6.90 ″10⁻² 1.8 1.13 0.99 1.06 3.18 ″ 10⁻¹ 1.1 0.80 0.97 0.97 2.74 ″ 1  1.00.01 0.22 0.13 0.36

EXAMPLE 52

[0313] A part of stem of Taxus baccata LINN which had been previouslysterilized with 2° C. antiformin solution or 70% ethanol solution andthe like, was placed on solid Woody Plant Medium (containing gelan gumof 0.25% by weight) to which naphthalenacetic acid had been added togive the concentration of 10⁻⁵M, and static culture was carried out at25° C. in a dark place to provide Taxus baccata LINN callus. One gram(fresh weight) of the callus was inoculated to an Erlenmeyer flaskcontaining 20 ml of liquid Woody Plant Medium to which theabove-mentioned component had been added to give the same concentrationand shake culture was carried out with a rotary shaker (amplification of25 mm, 100 rpm) and the callus was subcultured in every 21 days toaccelerate the growth rate thereof.

[0314] One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component had been added to give thesame concentration, and shake culture was carried out at 25° C. for 14days. Acetylsalicylic acid (HOOCC₆H₄OCOCH₃) was added as antiethyleneagent on the 14th day after starting the culture to give the finalconcentration of 10⁻⁹M-1M and the culture was further continued foranother 7 days.

[0315] After completing the culture, cultured cells of Taxus baccataLINN were harvested by filtration and lyophilized, then the dry weightwas measured to obtain the growth rate thereof. Taxane-type diterpeneswere extracted from the dried cells with methanol and the like, and theywere determined by comparing with standard taxol, cephalomannine, andbaccatin III using high performance liquid chromatography to measure theyields of the taxane-type diterpenes. The results are shown in Table 16.

EXAMPLE 53

[0316] The procedure of Example 52 was carried out except thatacetylsalicylic acid was added at the start (0 day) of the culture togive the final concentration of 10⁻⁵M and the culture was carried outfor another 21 days. After completing the culture, the procedure ofExample 52 was carried out. The results are shown in Table 16.

EXAMPLE 54

[0317] The procedure of Example 52 was carried out except thatacetylsalicylic acid was added on the 7th day after starting the cultureto give the final concentration of 10⁻⁵M and the culture was furthercontinued for another 14 days. After completing the culture, theprocedure of the Example 52 was carried out. The results are shown inTable 16.

EXAMPLE 55

[0318] The procedure of Example 52 was carried out except thatacetylsalicylic acid was added on the 18th day after starting theculture to give the final concentration of 10⁻⁵M and the culture wasfurther continued for another 3 days. After completing the culture, theprocedure of the Example 52 was carried out. The results are shown inTable 16.

EXAMPLE 56

[0319] The procedure of Example 52 was carried out except thatacetylsalicylic acid was added successively a total of 5 times at 2days' intervals starting from the 7th day after starting the culture (togive the final concentration of 2×10⁻⁶M each time, and to give the totalconcentration of 10⁻⁵M). The results are shown in Table 16.

EXAMPLE 57

[0320] The procedure of Example 52 was carried out except that methylester of jasmonic acid (which is a compound represented by the generalformula (III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f),R², R³, R⁴, R⁵, and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1and C³ and C⁴ have a double bond between them) was added on the 14th dayafter starting the culture to give the final concentration of 10⁻⁴M. Theresults are shown in Table 16.

EXAMPLE 58

[0321] The procedure of Example 52 was carried out, except that theflask was placed in a chamber (the capacity of 3000 ml) having a gasfeed opening and a gas discharge opening, then the chamber was closedhermetically, and air was so mixed with nitrogen that the concentrationof the oxygen in a gas to be supplied to the cells to be cultured became10%, and the gas was supplied through the feed opening at the rate of 25ml per minute. The results are shown in Table 16.

EXAMPLE 59

[0322] The procedure of Example 58 was carried out except that methylester of jasmonic acid was added on the 14th day of the culture to givethe final concentration of 10⁻⁴M. The results are shown in Table 16.

COMPARATIVE EXAMPLE 17

[0323] The procedure of Example 52 was carried out except thatacetylsalicylic acid was not added. The results are shown in Tables 16.

REFERENCE EXAMPLE 11

[0324] The procedure of Example 52 was carried out except that Ethrel(C₂H₆O₃ClP) of 10⁻³M was added instead of acetylsalicylic acid, as anethylene generating agent at the start (0 day) of the culture. Theresults are shown in Table 16.

Reference Example

[0325] The procedure of Example 52 was carried out except that Ethrel of10⁻³M was added instead of acetylsalicylic acid at the 14th day afterstarting the culture. The results are shown in Table 16.

EXAMPLE 60

[0326] The procedure of Example 52 was carried out except thataminoxyacetic acid hydrochloride [(H₂NOCH₂COOH)₂HCl] was added as anantiethylene agent to give the final concentration of 10⁻⁹M-1M. Theresults are shown in Table 17.

EXAMPLE 61

[0327] The procedure of Example 52 was carried out except that propylgallate [(HO)₃C₆H₂COOCH₂CH₂CH₃] was added as an antiethylene agent togive the final concentration of 10⁻⁹M-1M. The results are shown in Table18. TABLE 16 concentration concentration max oxygen ethylene yield^(a))yield^(a)) of yield^(a)) total yield^(b)) of of methyl concentrationconcen- growth of cephalo- of of acetylsalicylic jasmonate in the gastration rate baccatin mannine taxol taxane acid (M) (M) phase (%) (M)(times) III (mg/l) (mg/l) (mg/l) (mg/l) Comparative 0 0 20 0 2.7 0.140.56 0.28 0.98 Example 17 Example 52 10⁻⁹ 0 20 0 2.7 0.25 0.62 0.48 1.35″ 10⁻⁸ 0 20 0 2.8 0.79 0.64 0.89 2.32 ″ 10⁻⁷ 0 20 0 2.9 0.79 1.27 0.902.96 ″ 10⁻⁶ 0 20 0 2.5 0.83 2.73 3.00 6.56 ″ 10⁻⁵ 0 20 0 2.7 1.52 6.9613.42 21.90 ″ 10⁻⁴ 0 20 0 2.7 1.48 6.54 11.15 19.17 ″ 10⁻³ 0 20 0 2.61.06 3.30 4.44 8.80 ″ 10⁻² 0 20 0 2.6 1.01 0.70 1.61 3.32 ″ 10⁻¹ 0 20 02.0 0.58 0.12 0.70 1.40 ″ 1 0 20 0 1.5 0.01 0 0.01 0.02 Example 53 10⁻⁵0 20 0 2.6 0.42 1.76 2.98 5.16 Example 54 10⁻⁵ 0 20 0 2.7 0.91 3.55 4.228.68 Example 55 10⁻⁵ 0 20 0 2.7 1.22 6.39 9.58 17.19 Example 56 10⁻⁵ 020 0 2.6 1.09 5.46 10.02 16.57 Example 57 10⁻⁵ 10⁻⁴ 20 0 2.4 11.52 6.6015.11 33.23 Example 58 10⁻⁵ 0 10 0 2.4 7.28 6.46 11.80 25.54 Example 5910⁻⁵ 10⁻⁴ 10 0 2.3 14.79 6.69 18.91 40.39 Reference 0 0 20 10⁻³ 2.7 0.120.14 0.14 0.40 Example 1 Reference 0 0 20 10⁻³ 2.7 0.05 0.16 0.09 0.30Example 2

[0328] TABLE 17 concentra- yield^(a)) yield^(a)) total tion of of ofceph- yield^(a)) yield^(b)) aminoxy- growth baccatin aloman- of ofacetic acid rate III nine taxol taxane (M) (times) (mg/l) (mg/l) (mg/l)(mg/l) Comparative 0  2.7 0.14 0.56 0.28 0.98 Example 17 Example 60 10⁻⁹2.3 0.21 0.58 0.59 1.38 ″ 10⁻⁸ 1.8 0.61 0.62 0.77 2.00 ″ 10⁻⁷ 1.7 0.660.65 1.02 2.33 ″ 10⁻⁶ 1.5 1.08 0.73 1.19 3.00 ″ 10⁻⁵ 1.7 1.30 1.16 1.353.81 ″ 10⁻⁴ 1.7 1.92 1.22 2.66 5.80 ″ 10⁻³ 1.5 1.11 0.71 1.61 3.43 ″10⁻² 1.5 0.32 0.71 0.90 1.93 ″ 10⁻¹ 1.2 0.33 0.26 0.44 1.03 1  0.9 0 0 00

[0329] TABLE 18 concentra- yield^(a)) yield^(a)) total tion of of ofceph- yield^(a)) yield^(b)) propyl growth baccatin aloman- of of gallaterate III nine taxol taxane (M) (times) (mg/l) (mg/l) (mg/l) (mg/l)Comparative 0  2.7 0.14 0.56 0.28 0.98 Example 17 Example 61 10⁻⁹ 2.70.41 0.59 0.37 1.37 ″ 10⁻⁸ 2.7 0.42 0.68 0.74 1.74 ″ 10⁻⁷ 2.7 0.55 0.721.52 2.79 ″ 10⁻⁶ 3.1 1.91 4.38 7.66 13.95 ″ 10⁻⁵ 3.0 2.82 5.07 8.2416.13 ″ 10⁻⁴ 3.0 0.87 4.42 6.98 12.27 ″ 10⁻³ 3.2 0.84 4.30 6.96 12.10 ″10⁻² 2.9 0.69 2.01 4.33 7.03 ″ 10⁻¹ 1.9 0.48 0.25 0.65 1.38 ″ 1  1.10.01 0 0 0.01

EXAMPLE 62

[0330] A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵M, and static culture was carried out at 25° C.in a dark place to provide Taxus baccata LINN callus. One gram (freshweight) of the callus was inoculated to an Erlenmeyer flask containing20 ml of liquid Woody Plant Medium to which the above-mentionedcomponent had been added to give the same concentration and shakeculture was carried out with a rotary shaker (amplification of 25 mm,100 rpm) and the callus was subcultured in every 21 days to acceleratethe growth rate of the callus.

[0331] One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component had been added to give thesame concentration, and the flask was placed in a chamber (the capacityof 3000 ml) having a gas feed opening and a gas discharge opening, thenthe chamber was closed hermetically, and air was so mixed with nitrogenthat the concentration of the oxygen in a gas to be supplied to thecells to be cultured became 4-15%, and while the gas was suppliedthrough the feed opening at the rate of 25 ml per minute, shake culturewas carried out at 25° C. for 21 days.

[0332] After completing the culture, cultured cells of Taxus baccataLINN were harvested by filtration and lyophilized, then the dry weightwas measured to obtain the growth rate thereof. Taxane-type diterpeneswere extracted from the dried cells with methanol and the like, and theywere determined by comparing with standard taxol, cephalomannine, andbaccatin III using high performance liquid chromatography to measure theyields of the taxane-type diterpenes. The results are shown in Table 19.

COMPARATIVE EXAMPLE 18

[0333] The procedure of Example 62 was carried out except that theconcentration of the oxygen in a gas to be supplied to the cells to becultured was controlled to be 20%. The results are shown in Table 19.

REFERENCE EXAMPLE 3

[0334] The procedure of Example 62 was carried out except that thecultured cells inoculated to the flask were cultured in the atmosphere.The results are shown in Table 19.

EXAMPLE 63

[0335] The procedure of Example 62 was carried out except that theconcentration of the oxygen in a gas to be supplied to the cells to becultured was controlled to be 10% and the mixed gas was supplied fromthe start of the culture for 3 days, then air was supplied till the endof the culture (for 18 days). The results are shown in Table 19.

EXAMPLE 64

[0336] The procedure of Example 62 was carried out except that theconcentration of the oxygen in a gas to be supplied to the cells to becultured was controlled to be 10% and the mixed gas was supplied fromthe start of the culture for 7 days, then air was supplied till the endof the culture (for 14 days). The results are shown in Table 19.

EXAMPLE 65

[0337] The procedure of Example 62 was carried out except that theconcentration of the oxygen in a gas to be supplied to the cells to becultured was controlled to be 10%, and the mixed gas was supplied fromthe start of the culture for 14 days, then air was supplied till the endof the culture (for 7 days). The results are shown in Table 19.

EXAMPLE 66

[0338] The procedure of Example 62 was carried out except that methylester of jasmonic acid (which is a compound represented by the generalformula (III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f),R², R³, R⁴, R⁵, and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1and C³ and C⁴ have a double bond between them; 90% of which is in thetrans-form, and 10% of which is in the cis-form) was added as one ofjasmonic acids on the 14th day after starting the culture to give thefinal concentration of 10-1000 μM. The results are shown in Table 20.The productivity of taxane-type diterpene could be remarkably improvedby combination of supply of oxygen of low concentration and addition ofthe methyl ester of jasmonic acid.

EXAMPLE 67

[0339] Eighty-five gram (fresh weight) of cultured cells obtained inExample 62 with accelerated growth rate were inoculated in a tank forstirred tank culture (capacity of 3000 ml) having an electrode for adissolved oxygen concentration and a dissolved oxygen concentrationcontroller, into which 1700 ml of liquid Woody Plant Medium had beenpoured. Then stirred tank culture was carried out at 25° C. for 21 dayswhile the dissolved oxygen concentration in the medium was controlled tobe 0.1 ppm or less by adjusting the mixing ratio of air and nitrogen.The schematic diagram of the culture apparatus is shown in FIG. 3 andthe results are shown in Table 21.

EXAMPLE 68

[0340] The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen was controlled to be 1 ppm or lessby adjusting the mixing ratio. The results are shown in Table 21.

EXAMPLE 69

[0341] The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen was controlled to be 2 ppm or lessby adjusting the mixing ratio. The results are shown in Table 21.

EXAMPLE 70

[0342] The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen was controlled to be 4 ppm or lessby adjusting the mixing ratio. The results are shown in Table 21.

EXAMPLE 71

[0343] The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen was controlled-to be 6 ppm or lessby adjusting the mixing ratio. The results are shown in Table 21.

COMPARATIVE EXAMPLE 19

[0344] The procedure of Example 67 was carried out except that air wassupplied. The results are shown in Table 21.

EXAMPLE 72

[0345] The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen in the medium was controlled to be4 ppm or less from the start of the culture for 3 days by adjusting themixing ratio, and air was supplied till the end of the culture (for 18days). The results are shown in Table 21.

EXAMPLE 73

[0346] The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen in the medium was controlled to be4 ppm or less from the start of the culture for 7 days by adjusting themixing ratio, and air was supplied till the end of the culture (for 14days). The results are shown in Table 21.

EXAMPLE 74

[0347] The procedure of Example 67 was carried out, except that theconcentration of the dissolved oxygen in the medium was controlled to be4 ppm or less from the start of the culture for 14 days by adjusting themixing ratio, and air was supplied till the end of the culture (for 7days). The results are shown in Table 21. TABLE 19 max oxygen yield^(a))yield^(a)) total concentra- of of ceph- yield^(a)) yield^(b)) tion ingrowth baccatin aloman- of of the gas rate III nine taxol taxane phase %(times) (mg/l) (mg/l) (mg/l) (mg/l) Comparative 20 3.01 0.78 1.71 1.463.95 Example 18 Example 62  4 2.09 1.65 5.24 1.78 8.67 ″  7 2.60 3.377.25 2.49 13.11 ″ 10 2.90 3.19 7.51 2.96 13.66 ″ 15 2.82 3.18 6.86 2.1812.22 Example 63   10^(c)) 2.79 1.33 4.88 1.74 7.95 Example 64   10^(c))2.62 3.21 7.16 1.75 12.12 Example 65   10^(c)) 2.66 3.17 7.16 1.92 12.25Reference 20 3.03 0.77 1.23 0.95 2.95 Example 3

[0348] TABLE 20 yield^(a)) yield^(a)) total max oxygen concentration ofof yield^(b)) concentra- of methyl growth baccatin yield^(a)) cephalo-of tion in gas jasmonate rate III of taxol mannine taxane phase (%) (μM)(times) (mg/l) (mg/l) (mg/l) (mg/l) Comparative 20 0 3.01 0.78 1.71 1.463.95 Example 18 Example 62 10 0 2.90 3.19 7.51 2.96 13.66 Example 66 1010 2.82 2.26 10.24 1.73 14.73 ″ 10 100 2.45 11.32 22.44 2.29 36.05 ″ 101000 1.47 2.10 5.04 1.47 8.61

[0349] TABLE 21 max dissolved oxygen yield^(a)) yield^(a)) totalconcentra- of yield^(a)) of ceph- yield^(b)) tion in the growth baccatinof aloman- of medium rate III taxol nine taxane (ppm) (times) (mg/l)(mg/l) (mg/l) (mg/l) Comparative 8 (100^(c))) 3.01 0.58 1.74 0.91 3.23Example 19 Example 67 0.1 2.20 0.70 2.05 1.12 3.87 (1.25^(c))) Example68 1 (12.5^(c))) 2.25 1.05 3.69 1.22 5.96 Example 69 2 (25^(c))) 2.492.44 5.54 2.70 10.68 Example 70 4 (50^(c))) 2.75 3.91 8.14 1.80 13.85Example 71 6 (75^(c))) 2.98 2.52 5.69 1.23 9.44 Example 72 4^(d))(50^(e))) 2.89 1.62 4.02 1.54 7.18 Example 73 4^(d)) (50^(e))) 2.62 3.328.46 3.08 14.86 Example 74 4^(d)) (50^(e))) 2.75 3.19 7.72 2.65 13.56

EXAMPLE 75

[0350] A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10-5M, and static culture was carried out at 25° C.in a dark place to provide Taxus baccata LINN callus. One gram (freshweight) of the callus was inoculated to an Erlenmeyer flask containing20 ml of liquid Woody Plant Medium to which the above-mentionedcomponent had been added to give the same concentration and shakeculture was carried out with a rotary shaker (amplification of 25 mm,100 rpm), and the callus was subcultured in every 21 days to acceleratethe growth rate thereof.

[0351] One gram (fresh weight) of the cultured cells thus obtained wasfractionated firstly by a stainless steel mesh and cell clusters havingthe size of 250-840 μm were obtained. A density gradient with specificgravities of 1.03, 1.05, 1.07, 1.09 and 1.11 (g/ml) was produced by theuse of Ficoll and the above-mentioned cells were layered over it andcentrifuged at 700 rpm for 6 minutes. The cells were fractionated intoeach layer according to the difference of the specific gravity. Thecells contained in each layer were so fractionated that they were notmixed each other and washed with 2% sucrose solution three times ormore, to wash off Ficoll. After the washing, about 0.1 g (fresh weight)of the cells were transferred to a culture well having an inner diameterof 18 mm, containing 0.8 ml of liquid Woody Plant Medium and shakeculture was carried out at 25° C. for 21 days. After culturing them for21 days, the whole amount of the cells was transferred to a culture wellhaving an inner diameter of 36 mm containing 3 ml of the above-mentionedliquid medium and shake culture was further continued at 25° C. foranother 28 days.

[0352] After completing the culture, cultured cells of Taxus baccataLINN were harvested by filtration and lyophilized, then the dry weightwas measured to obtain the growth weight thereof per liter the liquidmedium. Taxane-type diterpenes were extracted from the dried callus withmethanol and the like, and they were determined by comparing withstandard taxol, cephalomannine, and baccatin III using high performanceliquid chromatography to measure the yields of the taxane-typediterpenes. The results are shown in Table 22, FIG. 4 and FIG. 5.

COMPARATIVE EXAMPLE 20

[0353] The procedure of Example 75 was carried out except thatfractionation according to the density gradient was not carried outafter separation of the cell clusters by the stainless mesh. The resultsare shown in Table 22, FIG. 4 and FIG. 5.

EXAMPLE 76

[0354] The procedure of Example 75 was carried out except that thecultured cells which have the same parent plant, but were induced tocallus in different stage were used. Provided that the cells containedin a layer having the specific gravity of 1.07 or more were collected inone group and cultured. The results are shown in Table 22.

COMPARATIVE EXAMPLE 21

[0355] The procedure of Example 76 was carried out except thatfractionation according to the density gradient was not carried outafter separation of the cell clusters by the stainless mesh. The resultsare shown in Table 22. TABLE 22 content*) content*) content*) specificcell of of of cepha- gravity yield baccatin taxol lomannine range (g/l)III (ppm) (ppm) (ppm) Comparative — 12.6 1.5 13.0 27.5 Example 20Example 75 <1.03  12.5 7.9 184.0 170.3 1.03-1.05 12.5 32.1 90.8 123.71.05-1.07 12.2 16.3 93.5 114.1 1.07-1.09 12.6 0.0 3.9 12.3 1.09-1.1111.9 0.0 2.1 8.6 1.11< 13.5 0.0 2.5 6.3 Comparative — 9.3 256.5 864.0662.5 Example 21 Example 76 1.03-1.05 9.0 151.3 1660.5 1050.2 1.05-1.079.9 155.7 1140.8 804.0 1.07< 9.5 19.0 333.1 161.3

REFERENCE EXAMPLE 4

[0356] About 0.2 g (fresh weight) of the cells obtained in Example 75which were cultured after being fractionated into a layer having thespecific gravity range of 1.03 or less (Table 22) were inoculated to aculture well having an inner diameter of 36 mm containing 3 ml of liquidWoody Plant Medium and shake culture was carried out at 25° C. foranother 28 days. After completing the culture, the cells were againfractionated by the density gradient with specific gravities of 1.03,1.05, 1.07, 1.09 and 1.11 (g/ml). Immediately after the density gradientfractionation, the cells were collected and the distribution of thefractionated cells and the contents of the taxane-type diterpenes weredetermined. The results are shown in Table 23, FIG. 6 and FIG. 7. TABLE23 content*) content*) specific distribution of content*) of cepha-gravity of cells baccatin of taxol lomannine range (%) III (ppm) (ppm)(ppm) Reference <1.03  20.2 13.9 123.9 66.5 Example 4 1.03-1.05 29.828.1 216.1 128.7 1.05-1.07 15.1 71.7 315.5 226.0 1.07-1.09 8.1 96.2475.8 382.6 1.09-1.11 6.2 123.5 619.4 511.8 1.11< 20.5 195.2 609.6 460.2

EXAMPLE 77

[0357] One gram (fresh weight) of the same cultured cells that were usedin Example 1 was inoculated to an Erlenmeyer flask containing 20 ml of aliquid containing 10⁻⁵M-10⁻²M of potassium peroxodisulfate and shaken at25° C. for 21 days to carry out the first stage of the culture.

[0358] After completing the culture, the cultured cells were harvestedby filtration and a part of the cells were used as seed cells for thesecond stage of the culture and the rest of the cells were subjected tothe measurement of the cell yield and the taxane content in the cells.Accordingly, 1 g (fresh weight) of the cultured cells were inoculated toan Erlenmeyer flask containing 20 ml of liquid Woody Plant Medium towhich naphthalenacetic acid had been added to give the concentration of10⁻⁵M and shake culture was carried out at 25° C. for 14 days. On the14th day of the culture, methyl jasmonate was added to the medium togive the concentration in the medium of 100 μM and the culture wasfurther continued for another 7 days. On the other hand, the rest of thecells obtained in the first stage of the culture were lyophilized, thenthe dry weight was measured to obtain the cell yield thereof per literof the liquid medium. The content of taxol in the dried cells wasmeasured by high performance liquid chromatography. The yield of thecells and the yield of taxol were measured for the cells obtained bysecond stage of the culture in the same manner as that for the cellsobtained in the first stage of the culture. The results are shown inTable 24.

COMPARATIVE EXAMPLE 22

[0359] The procedure of Example 77 was carried out except that potassiumperoxodisulfate was not used. The results are shown in Table 24. TABLE24 additives to be used in the first stage of the cultivation result ofthe first result of the second concentration of stage of the culturestage of the culture potassium yield of content of yield of yield ofperoxodisulfate time for cells taxol cells taxol in the medium addition(g/l) (ppm) (g/l) (mg/l) Example 77 10⁻⁵ M 1st day 17.1 97 15.6 14.0 ″10⁻⁴ M ″ 16.2 250 15.0 20.4 ″ 5 × 10⁻⁴ M ″ 15.5 183 14.4 16.3 ″ 10⁻³ M ″15.2 84 13.0 13.6 ″ 5 × 10⁻³ M ″ 14.0 45 12.1 11.7 Comparative — — 16.553 15.4 10.1 Example 22

EXAMPLE 78

[0360] A hundred grams (fresh weight) of the same cultured cells thatwere used in Example 1 were inoculated in a tank for stirred tankculture (capacity of 2 liter; FIG. 8) filled with 1 liter of standardliquid Woody Plant Medium (sucrose concentration: 20 g/l, nitrate ionconcentration: 14.7 mM, α-naphthalenacetic acid: 10⁻⁵M) to which 2 mM[Ag(S₂O₃)₂]³⁻ had been added, and the culture was started at 25° C. inthe dark at the agitation rate of 40 rpm, while air was fed at 0.1 literper minute, and a medium containing 20 g/l of sucrose and 20 mM ofsodium nitrate was supplied continuously in a period starting from thesecond day till the 14th day of the culture in such a manner that theamount of the sucrose added in one day became 2 g/l and the amount ofnitrate ion added in one day became 2 mmol/l, and the culture solutionwas continuously taken out through a discharge opening, which wasdifferent from the nutrition source feed opening, and to which astainless filter of 100 mesh was attached, at the same rate as that ofthe addition of the nutrition source solution (the medium renewing ratioin the culture vessel was 10% per day) to carry out stirred tank culturefor 21 days. After completing the culture, the cultured cells and themedium were collected and the yield of taxol was measured in the samemanner as that used in the said Example 1. The results are shown inTable 25.

COMPARATIVE EXAMPLE 23

[0361] The procedure of Example 78 was carried out except that thenutrition source was not added midway. The results are shown in Table25. TABLE 25 yield of cells yield of taxol (mg/l) (g dried weight/l)from cells from medium total Example 78 38 31 89 125 Comparative 22 3327  60 Example 23

EXAMPLE 79

[0362] Fifty grams (fresh weight) of the same cultured cells that wereused in Example 1, and 1 liter of liquid Woody Plant Medium weretransferred to a culture tank (capacity of 2 liter) and the culture wascarried out at 25° C. in the dark at the agitation rate of 40 rpm, atthe aeration speed of 0.01 liter per minute for 14 days. Theprecipitated cell volume (PCV) measured on the 14th day after startingthe culture was 0.2 liter. From the 14th day, supply of a fresh medium,wherein 3 mM [Ag(S₂O₃)₂]³⁻ had been added to the medium of the samecomposition as that of the initial medium, as well as taking out of aculture solution which was free from cells were started. The amount ofthe fresh medium to be supplied per day was ⅖ of the PCV at that timeand the amount of the culture solution free from the cells to be takenout was controlled to be 1 liter. On the 35th day after starting theculture, PCV reached 0.6 liter. After that, the stationary state wasmaintained by taking out the culture solution containing cells once aday to keep the average PCV to 0.6 liter and by taking out the- culturesolution free from cells to keep the amount of the culture solution to 1liter. The culture was carried out for 90 days after the start of theculture. The amount of the fresh medium supplied during the stationarystate of 60 days was 15 liters, the amount of the medium taken out ofthe culture tank was 14 liters and the amount of the cells obtained was0.15 kg (dry weight) and the specific growth rate μ was 0.08 (day⁻¹),the average medium renewing ratio was 2.88. The results of the analysisof cells and medium taken out from the culture tank under stationarystate showed that 525 mg of taxol was produced. That was equal to theproductivity of 8.8 mg/liter/day.

[0363] The amount of the taxol contained in the cells and the mediumwere measured in the same manner as that used in Example 1.

EXAMPLE 80

[0364] Fifty grams (fresh weight) of the same cultured cells that wereused in Example 1 and 1 liter of liquid Woody Plant Medium weretransferred to a culture tank (capacity of 2 liters) and the culture wascarried out in the dark, at the agitation rate of 40 rpm, at 25° C. for14 days, while an air to which 2% carbon dioxide gas had been added wasfed at 0.1 liter per minute. After completing the culture, the cells andthe medium were collected and 15.2 g of dry cells were obtained.Determination of the amount of taxol contained in the cells and in themedium which was carried out in the same manner as that used in Example1 showed that 31 mg of taxol was produced.

COMPARATIVE EXAMPLE 24

[0365] The procedure of Example 1 was carried out except that jasmonewas added instead of methyl tuberonate to give the final concentrationof 0.1-1000 μM. The results are shown in Table 26.

COMPARATIVE EXAMPLE 25

[0366] The procedure of Comparative Example 24 was carried out exceptthat jasmone was not added. The results are shown in Table 26. TABLE 26concentra- yield*) of yield*) yield*) of tion of cell baccatin ofcephalo- jasmone yield III taxol mannine (gM) (g/l) (mg/l) (mg/l) (mg/l)Comparative 0.1 12.2 0.3 3.0 1.2 Example 24 ″ 1 12.1 0.4 3.2 1.0 ″ 1011.3 0.3 3.3 0.8 ″ 100 11.3 0.3 3.2 0.6 ″ 1000 10.9 0.2 2.5 0.8Comparative 0 12.2 0.2 2.8 1.5 Example 25

Industrial Applicability

[0367] The present invention allows industrial production of ataxane-type diterpene including taxol which is useful as a therapeuticagent for ovarian cancer, mammary cancer, lung cancer and the like.

1. A method of producing a taxane-type diterpene wherein tissues orcells of a plant which produce taxane-type diterpenes are cultured inthe presence of at least one selected from the group consisting ofjasmonic acids, compounds containing a heavy metal, complex ionscontaining a heavy metal, heavy metal ions, amines and antiethyleneagents, then the taxane-type diterpenes are recovered from the resultingcultures.
 2. The method according to claim 1, wherein the culture iscarried out in the presence of jasmonic acids.
 3. The method accordingto claim 2, wherein jasmonic acids are compounds represented by thegeneral formula (I):

[wherein, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e) and R^(1f) respectivelyrepresent hydrogen atom, hydroxyl group, alkyl group having 1 to 6carbon atoms, or alkoxy group having 1 to 6 carbon atoms; R², R³, R⁴, R⁵and R^(6a) respectively represent hydrogen atom or alkyl group having 1to 6 carbon atoms; a side chain consisting of C¹—C²—C³—C⁴—C⁵—C⁶ maycontain one or more double bonds; R^(6b) represents hydroxyl group or—O— carbohydrate residue; R⁷ represents hydroxyl group, OM (wherein M isalkali metal atom, alkaline earth metal atom or NH₄), NHR⁸ (wherein R⁸represents hydrogen atom, acyl group having 1 to 6 carbon atoms, alkylgroup having 1 to 6 carbon atoms or amino acid residue), OR⁹ (wherein R⁹is alkyl group having 1 to 6 carbon atoms or carbohydrate residue), oralkyl group having 1 to 6 carbon atoms; n is an integer of 1-7; and inthe above-mentioned five-membered ring, a double bond may be formedbetween the neighboring member carbon atoms].
 4. The method according toclaim 3, wherein jasmonic acids represented by the general formula (I)are compounds represented by the general formula (I′):

[wherein, R¹ represents hydrogen atom or hydroxyl group; a side chainconsisting of C¹—C²—C³—C⁴—C⁵—C⁶ may contain a double bond between C¹ andC², between C² and C³, or between C₃ and C⁴; R^(6b) represents hydroxylgroup or —O— carbohydrate residue; R⁷ represents hydroxyl group, OM(wherein M is alkali metal atom, alkaline earth metal atom or NH₄),NHR^(8′) (wherein R^(8′) represents hydrogen atom, acyl group having 1to 4 carbon atoms, alkyl group having 1 to 4 carbon atoms or amino acidresidue) or OR^(9′) (wherein R^(9′) represents alkyl group having 1 to 4carbon atoms or carbohydrate residue); n is an integer of 1-7; and inthe above-mentioned five-membered ring, a double bond may be formedbetween the neighboring member carbon atoms].
 5. The method according toclaim 3, wherein a compound represented by the general formula (I) istuberonic acid or methyl tuberonate.
 6. The method according to claim 2,wherein jasmonic acids are compounds represented by the general formula(II):

[wherein, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e) and R^(1f) respectivelyrepresent hydrogen atom, hydroxyl group, alkyl group having 1 to 6carbon atoms, or alkoxy group having 1 to 6 carbon atoms; R², R³, R⁴, R⁵and R⁶ respectively represent hydrogen atom or alkyl group having 1 to 6carbon atoms; a side chain consisting of C¹—C²—C³—C⁴—C⁵—C⁶ may containone or more double bonds; R⁷ represents hydroxyl group, OM (wherein M isalkali metal atom, alkaline earth metal atom or NH₄), NHR⁸ (wherein R⁸represents hydrogen atom, acyl group having 1 to 6 carbon atoms, alkylgroup having 1 to 6 carbon atoms or amino acid residue), R⁹ (wherein R⁹is alkyl group having 1 to 6 carbon atoms or carbohydrate residue), oralkyl group having 1 to 6 carbon atoms; n is an integer of 1-7; and inthe above-mentioned five-membered ring, a double bond may be formedbetween the neighboring member carbon atoms].
 7. The method according toclaim 6, wherein jasmonic acids represented by the general formula (II)are compounds represented by the general formula (II′):

[wherein, R^(1′) represents hydrogen atom or hydroxyl group; a sidechain consisting of C¹—C²—C³—C⁴—C⁵—C⁶ may contain a double bond betweenC¹ and C², between C²and C³, or between C³ and c⁴; R^(7′) representshydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metalatom or NH₄), NHR^(8′) (wherein R^(8′) represents hydrogen atom, acylgroup having 1 to 4 carbon atoms, alkyl group having 1 to 4 carbon atomsor amino acid residue) or OR^(9′) (wherein R^(9′) represents alkyl grouphaving 1 to 4 carbon atoms or carbohydrate residue); n is an integer of1-7; and in the above-mentioned five-membered ring, a double bond may beformed between the neighboring member carbon atoms].
 8. The methodaccording to claim 6, wherein a compound represented by the generalformula (II) is cucurbic acid or methyl cucurbate.
 9. The methodaccording to claim 2, wherein jasmonic acids are compounds representedby the general formula (III):

[wherein, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e) and R^(1f) respectivelyrepresent hydrogen atom, hydroxyl group, alkyl group having 1 to 6carbon atoms, or alkoxy group having 1 to 6 carbon atoms; R², R³, R⁴, R⁵and R⁶ respectively represent hydrogen atom or alkyl group having 1 to 6carbon atoms; a side chain consisting of C¹—C²—C³—C⁴—C⁵—C⁶ may containone or more double bonds; R⁷ represents hydroxyl group, OM (wherein M isalkali metal atom, alkaline earth metal atom or NH₄), NHR⁸ (wherein R⁸represents hydrogen atom, acyl group having 1 to 6 carbon atoms, alkylgroup having 1 to 6 carbon atoms or amino acid residue), OR⁹ (wherein R⁹is alkyl group having 1 to 6 carbon atoms or carbohydrate residue), oralkyl group having 1 to 6 carbon atoms; n is an integer of 1-7; and inthe above-mentioned five-membered ring, a double bond may be formedbetween the neighboring member carbon atoms].
 10. The method accordingto claim 9, wherein jasmonic acids represented by the general formula(III) are compounds represented by the general formula (III′),

[wherein, R^(1′) represents hydrogen atom or hydroxyl group; a sidechain consisting of C¹—C²—C³—C⁴—C⁵—C⁶ may contain a double bond betweenC¹ and C², between C² and C³, or between C³ and C⁴; R^(7 ′) representshydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metalatom or NH₄), NHR^(8′) (wherein R^(8′) represents hydrogen atom, acylgroup having 1 to 4 carbon atoms, alkyl group having 1 to 4 carbon atomsor amino acid residue) or OR^(9′) (wherein R^(9′) represents alkyl grouphaving 1 to 4 carbon atoms or carbohydrate residue); n is an integer of1-7; and in the above-mentioned five-membered ring, a double bond may beformed between the neighboring member carbon atoms].
 11. The methodaccording to claim 9, wherein a compound represented by the generalformula (III) is jasmonic acid or methyl jasmonate.
 12. The methodaccording to claim 2, wherein the concentration of jasmonic acids in atissue culture medium is 0.01-1000 μM.
 13. The method according to claim2, wherein jasmonic acids are added when the cultured cells are in theexponential growth phase or in the stationary phase.
 14. The methodaccording to claim 2, wherein jasmonic acids are added to the culturemedium in a plurality of parts or continuously.
 15. The method accordingto claim 1, wherein the culture is carried out in the presence of atleast one selected from the group consisting of compounds containing aheavy metal, complex ions containing a heavy metal and heavy metal ions.16. The method according to claim 15, wherein the heavy metal is silver.17. The method according to claim 16, wherein the compounds containingsilver are at least one compound selected from the group consisting ofsilver nitrate and silver sulfate.
 18. The method according to claim 16,wherein the compounds containing silver are at least one compoundselected from the group consisting of silver fluoride, silver chlorate,silver perchlorate, silver acetate, silver sulfite, silverhexafluorophosphate(V), silver tetrafluoroborate, diamine silver(I)sulfate, and potassium diaminoargentate(I).
 19. The method according toclaim 16, wherein the complex ions containing silver are at least oneion selected from the group consisting of [Ag(S₂O₃)₂]³⁻ and[Ag(S₂O₃)₃]⁵⁻.
 20. The method according to claim 16, wherein the complexions containing silver are at least one ion selected from the groupconsisting of [Ag(NH₃)₂]⁺, [Ag(CN)₂]⁻, [Ag(CN)₃]²⁻, [Ag(SCN)₂]⁻, and[Ag(SCN)₄]³⁻.
 21. The method according to claim 16, wherein theconcentration of compounds containing silver, complex ions containingsilver or silver ion is 10⁻⁸M-10⁻¹M.
 22. The method according to claim16, wherein the concentration of compounds containing silver, complexions containing silver or silver ion is 10⁻⁷M-10⁻²M.
 23. The methodaccording to claim 15, wherein the heavy metal is cobalt.
 24. The methodaccording to claim 23, wherein the compounds containing cobalt are atleast one compound selected from the group consisting of cobaltchloride, cobalt nitrate and cobalt sulfate.
 25. The method according toclaim 23, wherein the compounds containing cobalt are at least onecompound selected from the group consisting of cobalt fluoride, cobaltperchlorate, cobalt bromide, cobalt iodide, cobalt selenate, cobaltthiocyanate, cobalt acetate, ammonium cobalt sulfate, cobalt(II)potassium sulfate, hexaamminecobalt(III) chloride,pentaammineaquacobalt(III) chloride, nitropentaamminecobalt(III)chloride, dichlorotetraamminecobalt(III) chloride hemihydrate,dinitrotetraamminecobalt(III) chloride, carbonatotetraamminecobalt(III)chloride, ammonium tetranitrodiamminecobaltate(III), sodiumhexanitrocobaltate( III), tris(ethylenediamine)cobalt( III) chloridetrihydrate, dichlorobis(ethylenediamine)cobalt(III) chloride, potassiumtris(oxalato)cobaltate(III) trihydrate, potassiumhexacyanocobaltate(III), potassium(ethylenediaminetetraacetato)cobaltate(III) dihydrate,hydridotetracarbonylcobalt(I), dicarbonyl(cyclopentadienyl)cobalt(I),octacarbonyldicobalt(O), hexacarbonyl(acetylene)dicobalt(O),bis(cyclopentadienyl)cobalt(I), and(cyclopentadienyl)(1,5-cyclooctadiene)cobalt(I).
 26. The methodaccording to claim 23, wherein the complex ions containing cobalt are atleast one ion selected from a group consisting of pentaammineaquacobaltion, nitropentaamminecobalt ion, dichlorotetraamminecobalt ion,dinitrotetraamminecobalt ion, carbonatotetraamminecobalt ion,tetranitrodiamminecobalt ion, hexanitrocobalt ion,tris(ethylenediamine)cobalt ion, dichlorobis(ethylenediamine)cobalt ion,tris(oxalato)cobalt ion, hexacyanocobalt ion, and(ethylenediaminetetraacetato)cobalt ion.
 27. The method according toclaim 23, wherein the concentration of compounds containing cobalt,complex-ions containing cobalt or cobalt ion is 10⁻⁵M-10⁻¹M.
 28. Themethod according to claim 23, wherein the concentration of compoundscontaining cobalt, complex ions containing cobalt or cobalt ion is 10⁻⁵M10⁻²M.
 29. The method according to claim 15, wherein culture is carriedout in the presence of jasmonic acids and at least one selected from thegroup consisting of compounds containing a heavy metal, complex ionscontaining a heavy metal and heavy metal ions.
 30. The method accordingto claim 1, wherein the culture is carried out in the presence ofamines.
 31. The method according to claim 30, wherein the amines arepolyamines.
 32. The method according to claim 31, wherein the polyaminesare at least one compound selected from the group consisting ofputrescine, cadaverine, spermidine, spermin, ethylene diamine,N,N-diethyl-1,3-propane diamine, diethylene triamine and a salt thereof.33. The method according to claim 31, wherein the concentration of thepolyamines is 10⁻⁸M-10⁻¹M.
 34. The method according to claim 31, whereinthe concentration of the polyamines is 10⁻⁷M-10⁻²M.
 35. The methodaccording to claim 30, wherein the culture is carried out in thepresence of amines and jasmonic acids.
 36. The method according to claim1, wherein the culture is carried out in the presence of an antiethyleneagent.
 37. The method according to claim 36, wherein the antiethyleneagent is a compound which inhibits the activity of an enzyme whichcatalyzes the conversion of S-adenosylmethionine into1-aminocyclopropane-1-carboxylic acid.
 38. The method according to claim37, wherein the antiethylene agent is at least one compound selectedfrom the group consisting of aminoxyacetic acid, acetylsalicylic acid,Rhizobitoxine, aminoethoxyvinylglycine, methoxyvinylglycine,α-aminoisobutyric acid, and a salt, an ester, an amino acid derivativeand a carbohydrate derivative thereof.
 39. The method according to claim36, wherein the antiethylene agent is a compound which inhibits theactivity of an enzyme which catalyzes the conversion of1-aminocyclopropane -1-carboxylic acid into ethylene.
 40. The methodaccording to claim 39, wherein the antiethylene agent is at least onecompound selected from the group consisting of gallic acid, and a salt,an ester, an amino acid derivative and a carbohydrate derivativethereof.
 41. The method according to claim 36, wherein the antiethyleneagent is a substance which removes the ethylene remaining in thecultures or existing in the gas phase or in the medium in the culturevessel containing the cultures.
 42. The method according to claim 41,wherein the antiethylene agent is at least one compound selected fromthe group consisting of 1,5-cyclooctadiene, and isothiocyanic acid, asalt, an ester, an amino acid derivative and a carbohydrate derivativethereof.
 43. The method according to claim 36, wherein the concentrationof the antiethylene agent is 10⁻⁸M-10⁻¹M.
 44. The method according toclaim 36, wherein the concentration of the antiethylene agent is10⁻⁷M-10⁻²M.
 45. The method according to claim 36, wherein the cultureis carried out in the presence of an antiethylene agent and jasmonicacids.
 46. The method according to claim 1, wherein the taxane-typediterpene is at least one compound selected from the group consisting oftaxol, 7-epitaxol, baccatin III, 7-epibaccatin III, cephalomannine,7-epicephalomannine, 10-deacetylbaccatin III, 10-deacetylcephalomannine,10-deacetyltaxol, taxagifine, xylosyl cephalomannine, and xylosyl taxol.47. The method according to claim 1, wherein the plant which producesthe taxane-type diterpene is a plant belonging to genus Taxus.
 48. Themethod according to claim 47, wherein the plant belonging to genus Taxusis at least one plant selected from the group consisting of Taxusbaccata LINN, Taxus cuspidata SIEB. et ZUCC, Taxus cuspidata SIEB. etZUCC var. nana REHDER, Taxus brevifolia NUTT, Taxus canadiensis MARSH,Taxus chinensis, and Taxus media.
 49. The method according to claim 1,wherein cells of the plant which produces the taxane-type diterpene arefractionated into a plurality of layers according to the difference intheir specific gravities, and cells contained in at least one layer arecultured.
 50. The method according to claim 1, wherein the tissues orthe cells of the plant which produce the taxane-type diterpene arecultured by controlling the oxygen concentration in a gas phase in aculture vessel to less than the oxygen concentration in the atmosphere,from the initial stage of the culture, or by controlling the dissolvedoxygen concentration in a fluid medium which is in contact with thetissues or the cells to less than the saturated dissolved oxygenconcentration at that temperature, from the initial stage of theculture.
 51. A method of producing a taxane-type diterpene whereintissues or cells of a plant which produce a taxane-type diterpene arecultured by carrying out a two-stage culture, comprising a first stageusing a medium to which an oxidizing agent or a water soluble organiccompound containing oxygen is added and a second stage which is carriedout according to the production method of claim 1, then the taxane-typediterpene is recovered from the resulting cultures.
 52. The methodaccording to claim 1, wherein the tissues or the cells of the plantwhich produce the taxane-type diterpene are cultured by inoculating thetissues or the cells in a culture medium containing a saccharide in aconcentration of 2-50 g/l, and/or nitrate ion in a concentration of 2-50mmol/l, then by adding a nutrient source solution containing thesaccharide in an amount of 0.2-5 g/l, and/or nitrate ion in an amount of0.2-5 mmol/l per day based on the initial volume of the said culturemedium, continuously or intermittently to the culture medium, then thetaxane-type diterpene is recovered from the resulting cultures.
 53. Themethod according to claim 52, wherein the culture is carried out whilethe culture medium is renewed by adding the nutrient source solution andseparating and removing the same volume of the culture medium from thetissues or the cells, and the taxane-type diterpene is recovered from atleast one substance selected from the resulting tissues and/or cells,the culture medium recovered during the culture and obtained at the endof the culture.
 54. The method according to claim 1, wherein a freshmedium is added continuously or intermittently in such a way that thespecific renewing ratio defined by the dimensionless number F=V_(I)/V/μ(wherein, V is the total volume of the culture solution in a culturetank, V_(I) is the feed speed of the fresh medium, and μ is the specificgrowth rate of the tissues or the cells) is in the range of 0.1 to 10,and the taxane-type diterpene is recovered from the culture medium andthe tissues or the cells contained in the culture medium which iscontinuously or intermittently taken out from the tank and/or from theculture medium containing no tissue and cell which is continuously orintermittently taken out from the tank.
 55. The method according toclaim 1, wherein the tissues or the cells of the plant which produce thetaxane-type diterpene are cultured by the use of oxygenic gas containing0.03-10% of carbon dioxide to be introduced to the culture vessel.
 56. Amethod of producing a taxane-type diterpene wherein tissues or cells ofa plant which produce a taxane-type diterpene are cultured bycontrolling the oxygen concentration in a gas phase in a culture vesselto less than the oxygen concentration in the atmosphere from the initialstage of the culture, or by controlling the dissolved oxygenconcentration in a fluid medium which is in contact with the tissues orthe cells to less than the saturated dissolved oxygen concentration atthat temperature from the initial stage of the culture, then thetaxane-type diterpene is recovered from the resulting cultures.
 57. Themethod according to claim 56, wherein the tissues or the cells of theplant which produce the taxane-type diterpene are cultured bycontrolling the oxygen concentration in the gas phase in the culturevessel to 4-15%, or by controlling the dissolved oxygen concentration inthe fluid medium which is in contact with the tissues or the cells, to1-75% of the saturated dissolved oxygen concentration at thattemperature.
 58. The method according to claim 56, wherein the tissuesor the cells of the plant which produce the taxane-type diterpene arecultured by controlling the oxygen concentration in the gas phase in theculture vessel to 6-12%, or by controlling the dissolved oxygenconcentration in the fluid medium which is in contact with the tissue orthe cell, to 10-75% of the saturated dissolved oxygen concentration atthat temperature.
 59. The method according to claim 56, wherein thetissue or the cell of the plant which produce the taxane-type diterpeneis cultured by controlling the oxygen concentration in the gas phase inthe culture vessel, or the dissolved oxygen concentration in the fluidmedium by adjusting the oxygen concentration in aeration gas to besupplied to the culture vessel and/or to the culture medium, or byadjusting the feed speed of the aeration gas to be supplied to theculture vessel and/or to the culture medium.
 60. The method according toclaim 56, wherein the controlling of the oxygen concentration in the gasphase in the culture vessel or the controlling of the dissolved oxygenconcentration in the fluid medium which is in contact with the tissuesor the cells is started between the beginning of the culture and the 7thday after the start of the culture and the controlling is kept at leastfor 3 days.
 61. The method according to claim 56, wherein thetaxane-type diterpene is at least one compound selected from the groupconsisting of taxol, 7-epitaxol, baccatin III, 7-epibaccatin III,cephalomannine, 7-epicephalomannine, 10-deacetylbaccatin III,10-deacetylcephalomannine, 10-deacetyltaxol, taxagifine, xylosylcephalomannine, and xylosyl taxol.
 62. The method according to claim 56,wherein the plant which produces the taxane-type diterpene is a plantbelonging to genus Taxus.
 63. The method according to claim 56, whereinthe culture is carried out in the presence of jasmonic acids.
 64. Amethod of producing a taxane-type diterpene wherein tissues or cells ofa plant which produce a taxane-type diterpene are cultured by carryingout a two-stage culture, comprising a first stage using a medium towhich an oxidizing agent or a water soluble organic compound containingoxygen is added and a second stage which is carried out according to theproduction method of claim 56, then the taxane-type diterpene isrecovered from the resulting cultures.
 65. The method according to claim56, wherein the tissues or the cells of the plant which produce thetaxane-type diterpene are cultured by inoculating the tissues or thecells in a culture medium containing a saccharide in a concentration of2-50 g/l, and/or nitrate ion in a concentration of 2-50 mmol/l, then byadding a nutrient source solution containing the saccharide in an amountof 0.2-5 g/l, and/or nitrate ion in an amount of 0.2-5 mmol/l per daybased on the initial volume of the said culture medium, continuously orintermittently to the culture medium, then the taxane-type diterpene isrecovered from the resulting cultures.
 66. The method according to claim65, wherein the culture is carried out while the culture medium isrenewed by adding the nutrient source solution and separating andremoving the same volume of the culture medium from the tissues or thecells, and the taxane-type diterpene is recovered from at least oneselected from the resulting tissues and/or cells, the culture mediumrecovered during the culture and obtained at the end of the culture. 67.The method according to claim 56, wherein a fresh medium is addedcontinuously or intermittently in such a way that the specific renewingratio defined by the dimensionless number F=V_(I)/V/μ (wherein, V is thetotal volume of the culture solution in a culture tank, V_(I) is thefeed speed of the fresh medium, and μ is the specific growth rate of thetissues or the cells) is in the range of 0.1 to 10, and the taxane-typediterpene is recovered from the culture medium and the tissues or thecells contained in the culture medium which is continuously orintermittently taken out from the tank and/or from the culture mediumcontaining no tissue and cell which is continuously or intermittentlytaken out from the tank.
 68. The method according to claim 56, whereinthe tissues or the cells of the plant which produce the taxane-typediterpene are cultured by the use of oxygenic gas containing 0.03-10% ofcarbon dioxide to be introduced to the culture vessel.
 69. A method ofproducing a taxane-type diterpene wherein tissues or cells of a plantwhich produce a taxane-type diterpene are cultured by the use ofoxygenic gas containing 0.03-10% of carbon dioxide to be introduced tothe culture vessel, and the taxane-type diterpene is recovered from theresulting cultures.
 70. A method of obtaining highly productive culturedcells for taxane-type diterpene, wherein cultured cells of a plant whichproduces a taxane-type diterpene are fractionated into a plurality oflayers according to the difference in their specific gravities, andcells contained in at least one layer are cultured, then highlyproductive cultured cells for the taxane-type diterpene are selectedfrom among those cultured cells.
 71. The method according to claim 70,wherein the plant which produces the taxane-type diterpene is a plantbelonging to genus Taxus.
 72. The method according to claim 70, whereincells contained in a layer having the specific gravity of 1.07 or lessare cultured.